Novel polypeptide hormone phosphatonin

ABSTRACT

The present invention relates to a novel human protein called phosphatonin, and isolated polynucleotides encoding this protein. Also provided are vectors, host cells, antibodies, and recombinant methods for producing this human protein. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating disorders related to this novel human protein.

FIELD OF THE INVENTION

[0001] The present invention relates to a polypeptide which is involvedin the regulation of phosphate metabolism. More specifically, thepresent invention relates to a novel polypeptide Metastatic-tumorExcreted Phosphaturic-Element (MEPE) or “phosphatonin”. This inventionalso relates to genes and polynucleotides encoding phosphatoninpolypeptides, as well as vectors, host cells, antibodies directed tophosphatonin polypeptides, and the recombinant methods for producing thesame. Also provided are diagnostic methods for detecting disordersrelating to phosphate metabolism, and therapeutic methods for treatingsuch disorders. The invention further relates to screening methods foridentifying agonists and antagonists of phosphatonin activity.

[0002] Several documents are cited throughout the text of thisspecification. Each of the documents cited herein (including anymanufacturer's specifications, instructions, etc.) are herebyincorporated herein by reference; however, there is no admission thatany document cited is indeed prior art as to the present invention.

BACKGROUND OF THE INVENTION

[0003] Phosphate plays a central role in many of the basic processesessential to the cell and the mineralization of bone. In particular,skeletal mineralization is dependent on the regulation of phosphate andcalcium in the body and any disturbances in phosphate-calciumhomeostasis can have severe repercussions on the integrity of bone. Inthe kidney, phosphate is lost passively into the glomerular filtrate andis actively reabsorbed via a sodium (Na+) dependent phosphatecotransporter. In the intestine, phosphate is absorbed from foods. Asodium (Na+) dependent phosphate cotransporter was found to be expressedin the intestine and recently cloned (Hilfiker, PNAS 95(24) (1998),14564-14569). The liver, skin and kidney are involved in the conversionof vitamin D3 to its active metabolite, calcitriol, which plays anactive role in the maintenance of phosphate balance and bonemineralization.

[0004] Vitamin D deficiency causes rickets in children and osteomalaciain adults. Both conditions are characterized by failure of calcificationof osteoid, which is the matrix of bone. There are also severalnon-dietary conditions which can lead to rickets, including X-linkedvitamin D resistant hypophosphatemic rickets (HYP), hereditaryhypercalciuria with hypophosphatemic rickets (HHRH), Dent's diseaseincluding certain types of renal Fanconi syndrome, renal Ialpha-hydroxylase deficiency (VDDR 1), defects in 1,25-dihydroxy vitaminD3 receptor (end organ resistance, VDDR II), and oncogenichypophosphatemic osteomalacia (OHO). Thus, a number of familial diseaseshave been characterized that result in disorders of phosphate uptake,vitamin D metabolism and bone mineralization. Recently a gene has beencloned and characterized that is defective in patients with X-linkedhypophosphatemic rickets (PHEX) (Francis, Nat. Genet. 11 (1995),130-136; Rowe, Hum. Genet. 97 (1996), 345-352; Rowe, Hum. Mol. Genet. 6(1997), 539-549). The PHEX gene is a type II glycoprotein and a memberof a family (M13), of Zn metalloendopeptidases. PHEX is proposed tofunction by processing a factor that plays a role in phosphatehomeostasis and skeletal mineralization (Rowe, Exp. Nephrol. 5 (1997),355-363; Rowe, Current Opinion in Nephrology & Hypertension 7(4) (1998),367-376). Oncogenic hypophosphatemic osteomalacia (OHO), has manysimilarities to HYP with an overlapping pathophysiology, but differentprimary defects (Rowe, Exp. Nephrol. 5 (1997), 355-363; Rowe, CurrentOpinion in Nephrology & Hypertension 7(4) (1998), 367-376; Drezner inPrimer on Metabolic Bone Diseases and Disorders of Mineral Metabolism(ed. Favus, M. J.) 184-188 (Am. Soc. Bone and Min. Res., Kelseyville,Calif., 1990)). Osteomalacia is the adult equivalent of rickets, and akey feature of tumour-acquired osteomalacia is softening of the bones.The softened bones become distorted, resulting in bow-legs and otherassociated changes reminiscent of familial rickets. Low serum phosphate,and abnormal vitamin D metabolism are also key features shared with HYP.Tumour acquired osteomalacia is rare, and the tumours are mainly ofmesenchyrnal origin, although a number of different tumour types havealso been reported (Rowe, Exp. Nephrol. 5 (1997), 355-363; Francis,Baillieres Clinical Endocrinology and metabolism 11 (1997), 145-163;loakimidis, The J. Rheumatology 21(6) (1994), 1162-1164; Lyles, Ann.Intem. Med. 93 (1980), 275-278; Rowe, Hum. Genet. 94 (1994), 457-467;Shane, Journal of Bone and Mineral Research 12 (1997), 1502-1511;Weidner, Cancer 59 (1987), 1442-1442). Surgical removal of the tumour(s)when possible, results in the disappearance of disease symptoms and bonehealing, suggesting the role of a circulating phosphaturic factor(s) inthe pathogenesis of the disease. Also, hetero-transplantation of tumoursinto nude mice (Miyauchi, J. Clin. Endocrinol. Metab. 67 (1988), 46-53)infusion of saline extracts into rats and dogs (Aschinberg, J. Paediatr.91 (1977), 56-60; Popovtzer, Clin. Res. 29 (1981), 418A (Abstract)), andthe use of tumour conditioned medium (TCM), of human and animal renalcell lines all confirm that a circulating phosphaturic factor issecreted by these tumours.

[0005] Although the primary-defect in X-linked rickets is confirmed as amutated Zn metalloendopeptidase (PHEX), there is considerable evidencethat implicates a circulating phosphaturic factor(s) (Ecarot, J. BoneMiner. Res. 7 (1992), 215-220; Ecarot, J. Bone Miner. Res. 10 (1995),424-431; Morgan, Arch. Intern. Med. 134 (1974), 549-552; Nesbitt, J.Clin. Invest. 89 (1992), 1453-1459; Nesbitt, J. Bone. Miner. Res. 10(1995), 1327-1333; Nesbitt, Endocrinology 137 (1996), 943-948; Qiu,Genet. Res., Camb. 62 (1993), 39-43; Lajeunesse, Kidney Int. 50 (1996),1531-1538; Meyer, J. Bone. Miner. Res. 4(4) (1989), 523-532; Meyer, J.Bone. Miner. Res. 4 (1989), 493-500). The overlapping pathophysiology ofHYP and OHO raises the intriguing possibility that the tumour-factor maybe processed in normal subjects by the PHEX gene product. Also, it islikely that proteolytic processing by PHEX may act by either degradingthis undefined phosphaturic factor(s), or by activating aphosphate-conserving cascade (Carpenter, Pediatric Clinics of NorthAmerica 44 (1997), 443-466; Econs, Am. J. Physiol. 273 (1997),F489-F498; Glorieux, Arch. Pediatr. 4 (1997), 102s-105s; Grieff, CurrentOpinion in Nephrology & Hypertension 6 (1997), 15-19; Hanna, CurrentTherapy in Endocrinology & Metabolism 6 (1997), 533-540; Kumar, Nephrol.Dial. Transplant. 12 (1997), 11-13; Takeda, Ryoikibetsu Shokogun Shirizu(1997), 656-659). The cloning and characterization of thetumour-phosphaturic factor is thus prerequisite to establishing anylinks between tumour osteomalacia and familial X-linked rickets as wellas other disorders in the phosphate metabolism.

[0006] Rowe et al (1996) have reported candidates 56 and 58 kDa protein(s) responsible for mediating renal defects in OHO (Rowe, Bone 18,(1996), 159-169). A patient with OHO was treated by tumor removal andpre- and post-operative antisera from the patient were used in a Westernblotting identification of tumor conditioned media proteins. Neither thetumor cells nor the antisera were ever made available to the public,however.

[0007] In a review in Exp. Nephrol. 5 (1997), 335-363, Rowe (1997)discusses the above diseases and the role of the PHEX gene (previouslyknown as the PEX gene). The PHEX gene product has been identified as azinc metalloproteinase. In disease states such as familial rickets,defective PHEX results in uncleaved phosphatonin which would result indown regulation of the sodium dependent phosphate cotransporter and upregulation of renal mitochondrial 24-hydroxylase. However, nopurification of phosphatonin was reported by Rowe (1997). Thus, nosource material for phosphatonin was made available to the public.Moreover, purification, identification and characterization ofphosphatonin has not been possible.

[0008] Thus, there is a need for polypeptides that regulate phosphatemetabolism, since disturbances of such a regulation may be involved inhypo- and hyperphosphatemic diseases, including osteomalacia,particularly osteoporosis and renal failure. Furthermore, there is aneed for identifying and characterizing such polypeptides which may playa role in the detection, prevention and/or correction of such disordersand may be useful in diagnosing those disorders.

SUMMARY OF THE INVENTION

[0009] The present invention relates to novel phosphatonin polypeptidesand the encoding polynucleotides of phosphatonin. Moreover, the presentinvention relates to vectors, host cells, antibodies, and recombinantmethods for producing the polypeptides and polynucleotides. Alsoprovided are diagnostic methods for detecting disorders related to thepolypeptides, and therapeutic methods for treating such disorders. Thepresent invention further relates to screening methods for identifyingbinding partners of phosphatonin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1: FIG. 1(a) and (b) show respectively chromatograms with lowaffinity and high affinity protein-containing peaks from a concanavalinA column.

[0011]FIG. 2: Cation exchange chromatogram of fractions from theconcanavalin A column.

[0012]FIG. 3: Computer prediction of hydrophilicity and hydrophobicityof phosphatonin.

[0013]FIG. 4: Computer prediction of antigenicity of phosphatonin.

[0014]FIG. 5: Computer prediction of flexibility of phosphatonin.

[0015]FIG. 6: Computer prediction of surface probability of thesecondary structure of phosphatonin.

[0016]FIG. 7: Computer prediction of the secondary structure ofphosphatonin.

[0017]FIG. 8: cDNA sequence (SEQ ID NO. 1) and amino acid sequence (SEQID NO: 2) of the largest MEPE clone isolated (PHO11.1). The five otherclone isolated are encompassed by this larger clone and all clone are inframe with the cloning vehicle pBSCPT SK II-. Primers used for PCR arehighlighted, and the total number of residues are 430 and 1655 bprespectively. The prokaryotic expression vector pCal-n-EK contained allin frame residues from MEPE residue V, to the MEPE stop codon (TAG), at1291-93 bp. The single polyadenylation sequence AA{T/U}AAA is doubleunderlined. The region of shared localized homology with DMA-1, DSSP,and OPN is underlined in wavy line format (MEPE-motif C-terminus), RGDresidues are enclosed in an ellipsoid), glycosaminoglycan attachmentsite is boxed (complete line format), Tyrosine Kinase site is underlinedonce, and N-glycosylation motifs are boxed in dotted line format. For acomplete list of motifs including casein kinase II, protein kinase Cetc. please refer to prosite screen Table 1.

[0018]FIG. 9: GCG-peptide-structure secondary structure prediction forMEPE. The primary amino acid backbone is shown as the central line withcurves indicating regions of predicted turn.Hydrophilicity/hydrophobicity regions are represented as ellipsoids anddiamonds respectively and the RGD motif is indicated. TheN-glycosylation sites are represented as ellipsoids on stalks(C-terminus), and alpha helix by undulating regions on the primarybackbone.

[0019]FIG. 10: Bar graphs showing phosphate-uptake in the presence ofdiffering amounts of MEPE: A. 92 ng/ml, B. 300 ng/ml, C. 500 ng/ml, andD. 1000 ng/ml. Choline boxes refer to control Na- independent resultswith NaCl replaced with choline chloride. Error bars are SEM, and Pvalues for the difference between MEPE and control in C and D are<0.001. In experiment A (92 ng/ml) P<0.05, and in B (300 ng/ml P, 0.01).N values for A and B are 4, and for C and D 5 and 6 respectively. Anovafollowed by Newman-Keuls Multiple Comparison Test was used.

[0020]FIG. 11: Dose curve of MEPE administration and phosphate uptakewith SEM error bars.

[0021]FIG. 12: Sequence similarity analysis using ‘sim’ and llanviewmathematical and software tools (Duret, Comput. Appl. Biosci. 12 (1996),507-510). In each computation the gap open penalty was set to 12, andgap extension penalty 4. Comparison matrix for A was ‘PAM40’, andBLOSUM62 for B and C respectively (see Duret, Comput. Appl. Biosci. 12(1996), 507-510; Huang, Comput. Appl. Biosci. 8 (1992), 155-165; Huang,Comput. Appl. Biosci. (1990) 6, 373-381). The similarity score thresholdwas 70% in A, and 40% in B and C respectively. The highlighted blocksshown on each protein scheme represent sequence homologies of >80% in A,and >62% in B and C. Note that in MEPE versus DSSP (A), there are fivehomology blocks in DSSP of >80% sequence similarity to a single motif inMEPE (DSSESSDSGSSSES). A similar sequence homology is also apparent forDMA-I and OPN versus MEPE (B and C) and the MEPE is a feature of allthree proteins.

[0022]FIG. 13: Dot matrix comparison of DSSP versus MEPE using Antheprotstatistical analysis (Deleague, G. Software for protein analysis:Antheroplot V2.5e. Microsoft group. (7 Passage du Vercours 69-367Vercors Lyon Cedex 07, 1997)). In (A) a lower stringency comparison witha window set to 13 is used as screen parameters and in (B) a widerwindow of 15 is used. The colors indicate unity matrix scores asindicated on the diagram. C-terminal residues of MEPE-motif have >80%sequence homology and the repeat nature of the motif is illustrated bythe striped pattern.

[0023]FIG. 14: p1BL21 and also p6XL1 recombinant plasmids containingphosphatonin fusion construct. LacI: (lac promoter); LIC: (Ligationindependent cloning sequence); EK: Enterokinase cleavage site; Thrombin(thrombin target sequence); Amp: Ampicillin resistance: Cal peptide(calmodulin peptide sequence); Phosphatonin (phosphatonin codingsequence).

[0024]FIG. 15: Primary structure of the entire phosphatonin molecule.Structural and functional motifs in the amino acid sequence (SEQ ID NO:27) of phosphatonin are indicated. For further explanations see text andlegend to FIG. 8 and Table 1.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0025] In view of the need of diagnostic and therapeutic means for thetreatment of diseases related to disorders in the phosphate metabolismin the human body, the technical problem of the invention is to providemeans and methods for the modulation of phosphate metabolism which areparticularly useful for the treatment of bone mineral and renaldiseases.

[0026] The above-defined technical problem is solved by the presentinvention by providing the embodiments characterized in the claims.Accordingly, in one aspect the present invention relates to an isolatedpolypeptide having phosphatonin activity.

[0027] Unless otherwise stated, the terms used herein are defined asdescribed in “A multilingual glossary of biotechnological terms: (IUPACRecommendations)”, Leuenberger, H. G. W., Nagel, B. and K÷lbl, H. eds.(1995), Helvetica Chimica Acta, CH-4010 Basle, Switzerland, ISBN 3-906390-13-6. The following definitions are provided to facilitateunderstanding of certain terms used throughout this specification.

[0028] The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacological and/or physiologicaleffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of partially or completely curing a disease and/oradverse effect attributed to the disease. The term “treatment” as usedherein covers any treatment of a disease in a mammal, particularly ahuman, and includes: (a) preventing the disease from occurring in asubject which may be predisposed to the disease but has not yet beendiagnosed as having it; (b) inhibiting the disease, i.e. arresting itsdevelopment; or (c) relieving the disease, i.e. causing regression ofthe disease. The present invention is directed towards treating patientswith medical conditions relating to a disorder of phosphate metabolism.Accordingly, a treatment of the invention would involve preventing,inhibiting or relieving any medical condition related to phosphatemetabolism disorders.

[0029] In the present invention, “isolated” refers to material removedfrom its original environment (e.g., the natural environment if it isnaturally occurring), and thus is altered “by the hand of man” from itsnatural state. For example, an isolated polynucleotide could be part ofa vector or a composition of matter, or could be contained within acell, and still be “isolated” because that vector, composition ofmatter, or particular cell is not the original environment of thepolynucleotide.

[0030] The phosphatonin polypeptide isolated in accordance with thepresent invention typically has an approximate molecular weight of 53 to60 kDa, more preferably 58-60 kDa, as measured on SDS-PAGE, particularlyon a 12.5% gel at pH 8.6 in TRIS-Glycine SDS buffer, see Example 1. Anapproximate molecular weight of 200 kDa may be measured onbis-tris-SDS-PAGE at pH 7 using a 4-12% gradient gel with MOPS runningbuffer. It is possible on such a gel also to see lower molecular weightbands of 53 to 60 kDa. The polypeptide is generally glycosylated, andpreferably comprises phosphatonin in substantially pure form.

[0031] Surprisingly, it has been found that the phosphatonin isobtainable, following purification according to the protocol given inExample 1 from Saos-2 cells, which are available from the EuropeanCollection of Cell Culture under Deposit No. ECACC 89050205.Accordingly, in a further aspect of the invention, there is provided useof Saos-2 cells or HTB-96 cells for the production of phosphatonin.Other transformed or immortalized cell lines may be capable ofoverexpression of phosphatonin, such as transformed osteoblast or bonecell lines.

[0032] The present invention also describes the characterization andcloning of a gene that is a candidate for the above-describedtumour-derived phosphaturic factor and that is named phosphatonin orMEPE (Metastatic-tumour Excreted Phosphaturic-Element). To summarize,expression screening of a λ ZAPII-cDNA library constructed from mRNAextracted from an OHO tumour using antisera specific to tumorconditioned media (TCM) phosphaturic-factor was used. The protein isglycosylated and resolves as two bands on SDS-PAGE electrophoresis(58-60 kDa), with evidence of possible splicing or post translationalcleavage. The first cloned cDNA codes for a protein of 430 residues (SEQID NO: 2) and 1655 bp in length (SEQ ID NO: 1). The entire 3′ end of thegene is present, with part of the 5′ end missing. Secondary structureprediction confirms that the protein is highly hydrophilic with smalllocalized regions of hydrophobicity and no cysteine residues. A numberof helical regions are present, with two distinct N-glycosylation motifsat the carboxy-terminus. A key feature is the presence of a cellattachment sequence in the same structural context found in osteopontin.Proteolytic-sites adjacent to this motif may result in altered receptorspecificity for specific integrins as found in osteopontin. Screening ofthe trembl database with MEPE sequence also demonstrated sequencehomology with Dentin phosphoryn (DPP). In particular there is strikinglocalized residue homology at the C′-terminus of MEPE with DPP,dentin-matrix protein-1 (DMA-1) and osteopontin (OPN). This region ofMEPE contains a recurring series of aspartate and serine residues(DDSSESSDSGSSSESD), with 80%, 65% and 62% homology with DSP, DMA-1 andOPN respectively. Moreover, when residue physicochemical character isconsidered this homology rises to 93%, suggesting a shared or relatedbiological-functionality. It is also of note that this structural motifoverlaps a casein kinase II phosphorylation motif in MEPE. Skeletalcasein kinase II activity is defective in rickets, and results in underphosphorylation of osteopontin (Rifas, Calcif. Tissue Int. 61 (1997),256-259). The casein kinase II defect has thus been proposed to play arole in the under-mineralization of bone matrix (Rifas, loc. cit.).

[0033] Dentin phosphoryn (DPP), is one part of a cleavage productderived from dentin sialophosphoprotein (DSSP), with the other partknown as dentin sialoprotein (DSP) (MacDougall, J. Biol. Chem. 272(1997), 835-842). It is of particular interest that DSSP, DMA-1, OPN andMEPE are RGD containing phospho-glycoproteins with distinct structuralsimilarities and major roles in bone-tooth mineralization (Linde, Crit.Rev. Oral Biol. Med. 4 (1993), 679-728).

[0034] The new OHO tumour-derived phosphaturic factor named phosphatoninor MEPE described in the present invention, effects bone mineralhomeostasis by regulating Na+ dependent phosphate co-transport, vitaminD metabolism, and bone mineralization.

[0035] As set out in further detail below, a polynucleotide has beenisolated which encodes polypeptides according to the present invention;see Example 2. The nucleotide and amino acid sequences of phosphatoninare set out in FIG. 8 (SEQ ID NO: 1 and SEQ ID NO: 2, respectively). Inthe continuous research efforts on phosphatonin, a furtherpolynucleotide has been isolated which encodes the N-terminus of thephosphatonin polypeptide; see Example 13. The nucleotide and amino acidsequences of the full-length phosphatonin protein are set out in SEQ IDNO: 26 and SEQ ID NO: 27, respectively. The amino acid sequencedescribed in SEQ ID NO: 27 differs from the one shown in FIG. 8 by anadditional 95 amino acid residues and a correction of the first valineto leucine on the N-terminus end of the amino acid sequence set out inFIG. 8 (SEQ ID NO: 2) (total length 525 vs. previous 430), and theN-terminus contains a signal peptide of about 17 amino acid residuespreceded by a consensus methionine start codon. Primer extension andNorthern blot analysis confirm that the nucleotide sequence shown in SEQID NO: 26 is 4 to 6 nucleotides short of the complete cDNA. Also thecorresponding extended N-terminal sequence contains two cysteines (theonly cysteines found in the entire phosphatonin molecule). One of thecysteines, at position 31 of SEQ ID NO: 27, is optimally placed for theformation of homodimers or heterodimers and the other is five residuesdownstream from the methionine start codon (at the edge of the signalpeptide). This indicates that after cleavage of signal peptide andrelease of phosphatonin into the extracellular milieu the N-terminus maybe involved in covalent protein-protein interactions. The truncated formof Rec-MEPE used in the experiments described in Example 12 lacks thisportion of the molecule (95 residues missing including signal peptideand the residue on the N-terminus end was valine instead of leucine)and, thus, is incapable of forming covalent cysteine-cysteineinter-protein bridges. Moreover, the region upstream of the truncatedrec-MEPE protein contains a proteolytic cleavage site that is acandidate for cleavage by PHEX, NEP and Nardilysin. The PHEX, NEPproteolytic site is at residue 46 of SEQ ID NO: 27 (KDNIG(FHHLG) andwould result in removal of a portion of the N-terminus containing thecysteine. It should be emphasized that these proteolytic-sites arehighly conserved and occur infrequently. The remaining molecule (missingcleaved N-terminus) is, thus, similar to the truncated form of rec-MEPE.The implication is that the rec-MEPE used in Example 12 cannot formdimers resulting in phosphate-retaining properties. Tumor derived MEPEis thus predicted to form dimers and interfere with renal-phosphatehandling resulting in inhibition of phosphate uptake.

[0036] In accordance with this additional data, obtained in accordancewith the present invention, shows that 24-hydroxylase expression (anenzyme involved in the catabolism of 1.25 (OH)2 vitamin D3, andup-regulated in X-linked rickets (Hyp)), is suppressed in a human renalcell-line exposed to rec-MEPE. Thus, both phosphate uptake and vitamin Dmetabolism expression are consistent with the above interpretation.Accordingly, the polypeptide of the present invention comprises theamino acid sequence of SEQ ID NO: 27, optionally including mutations ordeletions which do not substantially affect the activity thereof. Suchmutations include substitution of one or more amino acids, particularlyby homologues thereof, as well as additions of one or more amino acids,especially at the N or C termini. For example, a polymorphism at aminoacid residue 106 in the amino acid sequence of SEQ ID NO: 27 has beenfound where the glutamate can also be glycine. There appears to be anequal distribution in the clones thus far analyzed. Deletions includedeletions from the N or C termini. Substitutions by bothnaturally-occurring and synthetic amino acids are possible. Alsoincluded are polypeptides modified by chemical modification or enzymaticmodification. Further, fragment peptides, whether chemically synthesizedor produced by a biological method, whether modified or unmodified, areincluded within the scope of this invention.

[0037] Accordingly the present invention relates to a phosphatoninpolypeptide or an immunologically and/or biologically active fragmentthereof, which comprises an amino acid sequence encodable by apolynucleotide selected from the group consisting of

[0038] (a) polynucleotides encoding at least the mature form of thepolypeptide comprising the amino acid sequence depicted in SEQ ID NO: 2(FIG. 8) or SEQ ID NO: 27;

[0039] (b) polynucleotides comprising the coding sequence as depicted inSEQ ID NO: 1 (FIG. 8) or SEQ ID NO: 26 encoding at least the mature formof the polypeptide;

[0040] (c) polynucleotides comprising at least a nucleotide sequenceencoding amino acid residues 1 to 46, 1 to 96, 18 to 46, 18 to 96, 47 to96,47 to 525, and 18 to 525 of SEQ ID NO: 27;

[0041] (d) polynucleotides encoding a polypeptide derived from thepolypeptide encoded by a polynucleotide of (a), (b) or (c) by way ofsubstitution, deletion and/or addition of one or several amino acids ofthe amino acid sequence encoded by the polynucleotide of (a), (b) or(c);

[0042] (e) polynucleotides comprising the complementary strand whichhybridizes with a polynucleotide of any one of (a) to (d);

[0043] (f) polynucleotides encoding a polypeptide the sequence of whichhas an identity of 60% or more to the amino acid sequence of thepolypeptide encoded by a polynucleotide of any one of (a) to (e);

[0044] (g) polynucleotides encoding a polypeptide capable of regulatingphosphate metabolism comprising a fragment or an epitope-bearing portionof a polypeptide encoded by a polynucleotide of any one of (a) to (f);

[0045] (h) polynucleotides encoding an epitope-bearing portion of aphosphatonin polypeptide comprising amino acid residues from about 1 to40, 141 to 180 and/or 401 to 429 in SEQ ID NO: 2 (FIG. 8) and/or aminoacid residues from about 18 to 46 and/or 47 to 96 of SEQ ID NO: 27;

[0046] (i) polynucleotides comprising at least 15 nucleotides of apolynucleotide of any one of (a) to (h) and encoding a polypeptidecapable of regulating phosphate metabolism;

[0047] (j) polynucleotides encoding a polypeptide capable of regulatingphosphate metabolism comprising the cell and/or glycosaminoglycanattachment motif and/or the bone mineral motif of a polypeptide encodedby a polynucleotide of any one of (a) to (i);

[0048] (k) polynucleotides of any one of (a) to (j) wherein the encodedpolypeptide is capable of forming a homo- and/or heterodimer; and

[0049] (l) polynucleotides the nucleotide sequence of which isdegenerate as a result of the genetic code to a nucleotide sequence of apolynucleotide of any of (a) to (k).

[0050] As used herein, a phosphatonin “polynucleotide” refers to amolecule having a nucleic acid sequence contained in SEQ ID NO: 1 or 26or encoding the phosphatonin polypeptide of the present invention. Forexample, the phosphatonin polynucleotide can contain the nucleotidesequence of the full length cDNA sequence, including the 5′ and 3′untranslated sequences, the coding region, as well as fragments,epitopes, domains, and variants of the nucleic acid sequence. Moreover,as used herein, a phosphatonin “polypeptide” refers to a molecule havingthe translated amino acid sequence generated from the polynucleotide asbroadly defined.

[0051] A phosphatonin “polynucleotide” also includes thosepolynucleotides capable of hybridizing, under stringent hybridizationconditions, to sequences contained in SEQ ID NO: 1 or 26 or thecomplement thereof. “Stringent hybridization conditions” refers to anovernight incubation at 42 C in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20=g/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1× SSC atabout 65 C. Further suitable hybridization conditions are described inthe examples.

[0052] Also contemplated are nucleic acid molecules that hybridize tothe phosphatonin polynucleotides at lower stringency hybridizationconditions. Changes in the stringency of hybridization and signaldetection are primarily accomplished through the manipulation offormamide concentration (lower percentages of formamide result inlowered stringency); salt conditions, or temperature. For example, lowerstringency conditions include an overnight incubation at 37 C in asolution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA,pH 7.4), 0.5% SDS, 30% formamide, 100 (g/ml salmon sperm blocking DNA;followed by washes at 50 C with 1×SSPE, 0.1% SDS. In addition, toachieve even lower stringency, washes performed following stringenthybridization can be done at higher salt concentrations (e.g. 5× SSC).Note that variations in the above conditions may be accomplished throughthe inclusion and/or substitution of alternate blocking reagents used tosuppress background in hybridization experiments. Typical blockingreagents include Denhardt's reagent, BLOTTO, heparin, denatured salmonsperm DNA, and commercially available proprietary formulations. Theinclusion of specific blocking reagents may require modification of thehybridization conditions described above, due to problems withcompatibility. Of course, a polynucleotide which hybridizes only topolyA+sequences (such as any 3′ terminal polyA+ tract of a cDNA shown inthe sequence listing), or to a complementary stretch of T (or U)residues, would not be included in the definition of “polynucleotide,”since such a polynucleotide would hybridize to any nucleic acid moleculecontaining a poly (A) stretch or the complement thereof (e.g.,practically any double-stranded cDNA clone).

[0053] The phosphatonin polynucleotide can be composed of anypolyribonucleotide or polydeoxribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. For example, phosphatoninpolynucleotides can be composed of single- and double-stranded DNA, DNAthat is a mixture of single- and double-stranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. In addition, the phosphatoninpolynucleotides can be composed of triple-stranded regions comprisingRNA or DNA or both RNA and DNA. Phosphatonin polynucleotides may alsocontain one or more modified bases or DNA or RNA backbones modified forstability or for other reasons. “Modified” bases include, for example,tritylated bases and unusual bases such as inosine. A variety ofmodifications can be made to DNA and RNA; thus, “polynucleotide”embraces chemically, enzymatically, or metabolically modified forms.

[0054] Phosphatonin polypeptides can be composed of amino acids joinedto each other by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain amino acids other than the 20 gene-encodedamino acids. The phosphatonin polypeptides may be modified by eithernatural processes, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in the phosphatonin polypeptide,including the peptide backbone, the amino acid side-chains and the aminoor carboxyl termini. It will be appreciated that the same type ofmodification may be present in the same or varying degrees at severalsites in a given phosphatonin polypeptide. Also, a given phosphatoninpolypeptide may contain many types of modifications. Phosphatoninpolypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic phosphatonin polypeptides mayresult from posttranslation natural processes or may be made bysynthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivatives covalent attachment of a lipid or lipidderivative, covalent attachment of phosphatidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formulation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination;see, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed.,T. E. Creighton, W. H. Freeman and Company, New York (1993);POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson,Ed., Academic Press, New York (1983), pages. 1-12; Seifter, Meth.Enzymol. 182 (1990); 626-646, Rattan, Ann. NY Acad. Sci. 663 (1992);48-62. For example, it is possible that phosphatonin is expressed as apreproprotein and after processing of the pre-sequence and optionallypro-sequence is cleaved into two or more fragments which remain togetherdue to the formation of, for example, hydrogen bonds and/or disulfidebridges. The processing and/or cleavage of the prepro- and even matureform of the phosphatonin polypeptide may be accompanied by the loss ofone or more amino acids at the cleavage site. It is to be understoodthat all such forms of the phosphatonin protein are encompassed by theterm “phosphatonin polypeptide”, “polypeptide” or “protein”.

[0055] “SEQ ID NO: 1” and “SEQ ID NO: 26” refer to a phosphatoninpolynucleotide sequence while “SEQ ID NO: 2” and “SEQ ID NO: 27” referto a phosphatonin polypeptide sequence.

[0056] A phosphatonin polypeptide “having biological activity” refers topolypeptides exhibiting activity similar, but not necessarily identicalto, an activity of a phosphatonin polypeptide as measured in aparticular biological assay such as described below, with or withoutdose dependency. In the case where dose dependency does exist, it neednot be identical to that of the phosphatonin polypeptide, but rathersubstantially similar to the dose-dependence in a given activity ascompared to the phosphatonin polypeptide (i.e., the candidatepolypeptide will exhibit greater activity or not more than about 25-foldless and, preferably, not more than about ten-fold less activity, andmost preferably, not more than about three-fold less activity relativeto the phosphatonin polypeptide).

[0057] The term “immunologically active” or “immunological activity”refers to fragments, analogues and derivatives of the phosphatoninpolypeptide of the invention the essential characteristic immunologicalproperties of which remain unaffected in kind, that is that thepolynucleotides of the invention include all nucleotide sequencesencoding proteins or peptides which have at least a part of the primaryand/or secondary structural conformation for one or more epitopescapable of reacting specifically with antibodies unique to phosphatoninproteins which are encodable by a polynucleotide as set forth above.Preferably, the peptides and proteins encoded by a polynucleotide of theinvention are recognized by an antibody that specifically reacts with anepitope of the phosphatonin polypeptide comprising the amino acidresidues of about20to30, 100 to 130, 145 to 160, 300to310,320to340or380to430of SEQ ID NO: 2or with an epitope of the phosphatoninpolypeptides described herein below. Residues 380-430peptides/antibodies are particularly useful for the study ofmineralization processes, residues 145-160 peptides/antibodies for thestudy of receptor ligand interactions (inter gins etc.) and residues20-30 and 100-130, are of particular interest for phosphate regulationsstudies. Further preferred epitopes that are present in the phosphatoninpolypeptide of the present invention comprise amino acid residues 1 to46, 1 to 96, 18-46, 18 to 96, 47-96, 47 to 525, and/or 18-525 of SEQ IDNO: 27. In a particular preferred embodiment, the phosphatonin peptideof the present invention comprises the amino acid sequence from aminoacid position 18 to 46, 47 to 525 of SEQ ID NO: 27.

[0058] Preferably, the immunologically active phosphatonin peptidefragments, analogues and derivatives of the phosphatonin polypeptide ofthe invention are capable of eliciting an immune response in a mammal,preferably in mouse or rat.

[0059] In a preferred embodiment of the present invention thephosphatonin polypeptide is biologically active in that it is capable ofregulating or modulating phosphate metabolism, preferably it has“phosphatonin activity”.

[0060] Phosphatonin Activity

[0061] The term “capable of regulating or modulating phosphatemetabolism” as used herein means that the presence or absence, i.e. thelevel of the phosphatonin polypeptide of the invention in a subjectmodulates Na+-dependent phosphate co-transport, vitamin D metabolismand/or bone mineralization. Depending on whether the mentionedactivities are up- or down-regulated by the polypeptide of theinvention, said “capability of regulating or modulating phosphatemetabolism” is referred to as “phosphatonin activity” and“anti-phosphatonin activity”, respectively.

[0062] Phosphatonin activity may be measured by routine assay,particularly as the ability to down-regulate sodium dependent phosphateco-transport and/or up-regulate renal 25-hydroxy vitaminD3-24-hydroxylase and/or down-regulate renal 25-hydroxy-D-1(-hydroxylase. In each case, regulation of the relevant enzyme activitymay be effected directly or indirectly by the phosphatonin; e.g., bymeasurement of radioactive Na-dependent uptake of phosphate. Theseactivities may be assayed using a suitable renal cell line such as CL8or OK (deposited at the European Collection of Cell Cultures under ECACC91021202). A suitable assay methodology is found in Rowe et al (1996).Phosphatonin activity may further be measured by the ability to promoteosteoblast-mediated mineralization in tissue culture; see, e.g.,Santibanez, Br. J Cancer 74 (1996), 418-422; Stringa, Bone 16 (1995),663-670; Aronow, J. Cell Physiol. 143 (1990), 213-221; or as describedin the appended examples.

[0063] In a further aspect, the present invention provides a polypeptidecomprising a bioactive fragment of the polypeptide described above.Without intending to be bound by theory, it is thought that phosphatoninmay function as a polyhormone which may be cleaved in vivo to form oneor more fragments at least some of which possess biological activitysuch as hormonal activity. In vivo it is thought that phosphatonin maybe cleaved proteolytically, for example by the PHEX gene product toproduce at least one functional fragment. In a preferred embodiment, thepolypeptide comprising the bioactive fragment is capable of regulatingphosphate metabolism, for example by possessing phosphatonin activity asdiscussed above, or by possessing the opposite of phosphatonin activityas discussed in further detail below. The bioactive fragment may be anN-terminal, C-terminal or internal fragment. The polypeptide comprisingthe bioactive fragment may further comprise additionally amino acidsequence provided that the activity of the bioactive fragment is notsubstantially affected.

[0064] Advantageously, the bioactive fragment has a cell attachmentmotif which preferably comprises RGD. As discussed in further detailbelow, this motif may be involved in receptor and/or bone mineral matrixinteraction. Advantageously, the bioactive fragment has aglycosaminoglycan attachment motif, which preferably comprises SGDG (SEQID NO: 3). Attachment of glycosaminoglycan is thought to permit thefragment to resemble a proteoglycan. Proteoglycans are known to beinvolved in bone bioactivity, particularly in cell signaling. Thesemotifs are discussed in greater detail below. The primary structure ofthe entire phosphatonin molecule including structural and functionalmotifs is shown in FIG. 15. Any bioactive and/or immunologically activefragment of phosphatonin can be generated therefrom by recombinant DNAtechnology and/or (bio)chemical means, for example subjecting thephosphatonin molecule or derivatives thereof to enzymes such asendopeptidase and phosphatase in vitro or in vivo.

[0065] In one embodiment of the present invention, the polypeptidecomprising the bioactive fragment possesses phosphatonin activity:Without intending to be bound by theory, such activity is expected inphosphatonin uncleaved by PHEX metalloproteinase and some bioactivefragments carrying a PHEX metalloproteinase cleavage site such as thesite ADAVDVS (SEQ ID NO: 4) where cleavage is proposed to occur betweenresidues VD (residues 235 and 236 in the amino acid sequence of FIG. 8corresponding to position 330 and 331 of SEQ ID NO: 27) and/or betweenresidues GF at position 46 and 47 in the amino acid sequence of SEQ IDNO:. 27. The bioactive fragment may comprise at least the first 236residues of the amino acid sequence of FIG. 8 so that this PHEXmetalloproteinase cleavage site is part of the fragment. Suchpolypeptides and fragments thereof having phosphatonin activity will beuseful in treating hyperphosphatemic conditions.

[0066] It is to be understood that with regards to PHEX specificity inaccordance with the present invention preferably a tighter definition isused that includes glycine and phenylalanine (GF) as conserved and goodcandidate sites for cleavage. In this regard there are only three suchsites in the entire phosphatonin molecule at amino acidresidue-positions (46, 125, and 283 in SEQ ID NO: 27); see also FIG. 15.

[0067] Also the bioactive fragment(s) may include the entire moleculedownstream from the first PHEX site comprising of 479 residues of SEQ IDNO: 27 and the uncleaved molecule minus the putative signal peptide 508residues (particularly with regards to phosphate uptake). In this regardit is proposed that the cysteine (residue 31), N-terminal to theoriginal truncated rec-MEPE is prerequisite for the formation ofPhosphatonin homodimers and/or heterodimers.

[0068] Again, without intending to be bound by theory it is believedthat uncleaved dimerised phosphatonin is associated with phoshatoninactivity (renal phosphate leak, phosphaturia, down-regulation of Na+dependent phosphate co-transporters, and increase in 24-hydroxylaseactivity, hypophosphataemia etc.). Undimerised N-terminal cleavedphosphatonin is proposed to be associated with anti-phosphatoninactivity, (increase in renal phosphate uptake, up-regulation of Na+dependent phosphate co-transporters, and suppression of 24-hydroxylase,hyperphosphataemic and increase in 1-a-hydroxylase activity etc.).

[0069] With regard to bone mineralisation, it is surmised that theC-terminal region containing the MEPE-motif (probably afterphosphorylation) interacts with the bone matrix (hydroxyapatite,collagen type I, fibronectin, osteocalcin), and the RGD motif interactswith cells via integrin receptors (osteoblasts and/or osteoclasts). Thematrix cell interaction may play a key role in the nuclear-mineralsationand phosphatonin may also have an autocrine or paracrine effect onosteoblasts/osteoclasts gene-regulation and renal endocrine function.

[0070] Another possible scenario is the cleavage of phosphatonin intodefined segments that each has specific bioactivity (as occurs with anumber of hormones and bone morphogenetic proteins). Moreover,post-translational modification via phosphorylation may also play a keyrole in modulating phosphatonin bioactivity. For example, a fragment(158 residues) encompassing the RGD motif could theoretically begenerated by PHEX cleavage in the phosphatonin molecule at residues 125and 283 of SEQ ID NO: 27 and this may have defined bioactivity. AnN-terminal fragment generated by cleavage at residues 46 and 125 (79residues) in SEQ ID NO: 27 may also play a role in phosphatehomeostasis, and the possibility of a C-terminal fragment of 242residues generated by cleavage at residue 283 in the amino acid sequenceof SEQ ID NO: 27 playing a role in bone-mineralisation is envisaged aswell.

[0071] From the above observations and facts it is reasonable to proposethat pro-phosphatonin on conversion to phosphatonin (signal peptidecleavage) is cleaved by PHEX or other endopeptidases resulting in a lossof the cysteine-containing N-terminal peptide. Homodimers orheterodimers of the remaining C-terminal protein would thus not bepossible (no other cysteines in the entire phosphatonin-molecule).Formation of phosphatonin dimers may result in defective renal phosphatehandling by blocking renal NPT2 receptors (competing with processedphosphatonin), down-regulating NPT2 expression, affecting NPT2 mRNAstability, or altering intravesicular mobilisation of Na+ dependentphosphate co-transporters (either directly or indirectly). Removal ofthe N-terminal cysteine-containing region by PHEX or otherendopeptidases would result in a truncated or processed form of themolecule with phosphate retaining activity. The recombinantfusion-protein used in the experiments of Example 12 lacks theN-terminal “cysteine-containing” region (N-terminal 95 residues of SEQID NO: 27 are missing and the leucine residue next to such 95 residueswas valine) and may therefore act by imitating the phosphate-conservingor processed-form of MEPE (PHEX or metalloendopeptidase cleaved).Relevant to the presence of a unique zinc endopeptidase cleavage site inthe N-terminal MEPE region for PHEX/NEP and the proposed dimerisationmodel, is the presence of a single site for proprotein convertases I andII. The regulation of bone morphogenetic protein (BMP) activity and anumber of other hormones activities (pro-melanin, profibrillin,prothyrotropin-releasing hormone) by proprotein-convertases (PACE4 etc.)via endoproteolytic-cleavage of inactive precursor proteins has beendemonstrated (Constam, J. Cell Biol. 144 (1999), 139-149; Raghunath, J.Cell Sci. 112 (1999), 1093-1100; Viale, J. Biol. Chem. 274 (1999),6536-6545; Schaner, J. Biol. Chem. 272A (1997), 19958-19968). Also, theexpression pattern of specific pro-convertases during embryogenesis aredynamic and colocalisation with BMP's occurs (Tsuji, J. Biochem. 126(1999), 591-603).

[0072] In summary, it is believed that MEPE or phosphatonin issequentially processed by proteolytic cleavage and optionallyphosphorylation to generate a number of bioactive peptides. Removal ofthe first 46 residues appears to alter phosphatonin activity toanti-phosphatonin activity (phosphatonin defined as hypophosphataemicand anti-phosphatonin as hyperphosphataemic). PHEX is likely to play akey role in this process but the possibility of the involvement of otherproteases is not excluded. For example the proprotein convertases havetwo possible cleavage sites at residues 54 and 383 in the amino acidsequence of SEQ ID NO: 27 and could generate a much larger RGDcontaining peptide (329 residues). There may also be alternativesplicing and there is some evidence that suggests that the phosphatonintranscript in bone-marrow is of a different size to that found in theOHO-tumours.

[0073] Related Proteins

[0074] Further studies carried out in accordance with the presentinvention revealed a number of distinct similarities betweenphosphatonin (MEPE), dentin matrix protein-1 (DMP1), dentin sialophosphoprotein (DSSP; more specifically the dentin phosphorynC-terminus), bone sialoprotein (BSP) and osteopontin (OPN). Inparticular all the aforementioned matrix proteins have RGD motifs, areglycosylated with unusually high aspartate and serine contents. Caseinkinase II phosphorylation motifs are a common feature and there arelocalized regions of homology shared between each of the proteins.Lanview-sim analyses Swissprot software (Duret, LALNVIEW: a graphicalviewer for pairwise sequence alignments. Comput. Biosci. 12 (1996),507-510) graphically illustrate the regions of high homology as dotmatrix comparisons between phosphatonin and DSSP. The motif is repeatedfive times in the dentin phosphoryn (DP) portion of DSSP (FIG. 12a), andthis motif has 80% homology to a C-terninal residue in phosphatonin.Based on physiochemical parameters a 93% homology can be deduced andthis sequence homologue is present in the other bone/dentin moleculesdescribed with 60% to 65% sequence similarity. There is also in the sameregion extended sequence homology with a run of residues between DMA-1and phosphatonin as is shown in Table 2 and in the sequence comparisonbelow: 408 SSRRRDDSSESSDSGSSSESDG 429 MEPE (SEQ ID NO:5) 443SSRSKEDSN-STESKSSSEEDG 463 DMK-1 (SEQ ID NO:6)

[0075] Dentin sialo-phosphoprotein (DSSP) is a large RGD-containingglycoprotein that in-vivo is cleaved to generate two proteins known asdentin sialoprotein (DSP) and dentin phosphoryn (DP), respectively(MacDougall, J. Biol. Chem. 272 (1997), 853-842). DSP is the N-terminalpeptide and DP the C-terninal and both were originally thought to bederivatives of different genes. A statistical dot-matrix comparison ofphosphatonin versus DSSP at high and low stringency comparison is shownin FIG. 13. The repeat nature of the “motif-homologue” (DSSESSDSGSSSES(SEQ ID NO: 7)) in DSSP and its striking homology is clearly displayedin both graphical presentations. The motif is present only once in MEPEat the C-terminus. Moreover, overall low level sequence-similarity tothe C-terminal portion of DSSP (or the DP component) is clearlydisplayed. It is thus believed that a novel “unique” feature has nowbeen discovered that is likely to play a role in bone-mineralinteractions in bone-tooth matrix class of proteins.

[0076] In conclusion, all the proteins discussed appear to form integralassociations with bone mineral or tooth extracellular matrix and theinteractions are thought to be mediated via integrin/RGD associations.Moreover, the new regional motif (rich in serines and aspartate) wouldbe ideal for phosphate calcium interactions. This therefore supports thehypothesis that the C-terninus of phosphatonin plays a role in bonemineral homeostasis, and the N-terminus on renal phosphate regulation.In summary, the shared features of the proteins comprise:

[0077] 1. RGD motif in similar structural context.

[0078] 2. Glycoproteins.

[0079] 3. Rich in aspartate and serine.

[0080] 4. Casein kinase and protein kinase motifs.

[0081] 5. Distinct aspartate-serine rich MEPE motif (repeated in DPP).

[0082] 6. Large number of phosphorylation motif and myristoylationmotifs.

[0083] 7. Evidence of cleavage and/or alternative splicing.

[0084] 8. All associated with bone or tooth extracellular matrix.

[0085] Thus, in a preferred embodiment of the present invention, thephosphatonin polypeptide comprises the above-described bone mineralmotif, preferably the amino acid sequence of SEQ ID NO: 5 or 7 or anamino acid sequence corresponding to the same such as those from thementioned DMP1, DSSP, BSP, OPN or DMA-1 proteins.

[0086] Bioactive Fragments

[0087] In another embodiment of the present invention, the polypeptidecomprising the bioactive fragment has the reverse of phosphatoninactivity and may be suitable for treating hypophosphatemic conditions.In this embodiment, the polypeptide is directly or indirectly capable ofup-regulating sodium dependent phosphate cotransport and/ordown-regulating 25-hydroxy vitamin D3-24-hydroxylase and/orup-regulating renal 25-hydroxy-D-1(-hydroxylase. The mentionedactivities will also be referred to herein as “anti-phosphatonin”activity. However, use of the term “anti-phosphatonin” activity does notexclude the possibility that said activity is the one which ispredominant of genuine phosphatonin in phosphate metabolism. These“anti-phosphatonin” activities are also readily measurable using themethodology of Rowe et al (1996) by assay using a suitable renal cellline such as CL8 or OK (deposited at the European Collection of CellCultures under ECACC 91021202); see also the methods referred to supraand in the appended examples. Thus, the phosphatonin polypeptides of theinvention can be easily tested for phosphatonin or “anti-phosphatonin”activity according to any one of the methods referred to above ordescribed further herein, e.g., in the appended examples; see, e.g.,Example 12. Preferably, the fragment is obtainable by proteolyticcleavage of phosphatonin by a PHEX metallopeptidase. A PHEX gene hasbeen cloned and found to encode a zinc metalloendopeptidase as discussedin Rowe (1997). Again, without intending to be bound by theory,structurally, bioactive fragments having these activities are thought tolack at least a part of the N or C terminal portion of the amino acidsequence of FIG. 8 or FIG. 15, preferably lacking the N and/or Cterminal portion up to at least the putative PHEX metalloproteinasecleavage site at residues 235/236 in the amino acid sequence of SEQ IDNO: 2 or at residues 46, 125 and/or 283 of SEQ ID NO: 27. Thispolypeptide therefore preferably comprises no more than approximatelythe first 235 residues of the amino acid sequence of FIG. 8 or afragment of the amino acid sequence of SEQ ID NO: 27 generated by PHEXcleavage at any one the mentioned amino acid positions. Naturally,further modifications of such fragments such as those described aboveare included within the scope of the present invention.

[0088] As is explained in Example 4, the phosphatonin polypeptide of theinvention was cloned via the use of an expression library, wherein thetarget cDNA is fused to a portion of the (-galactosidase enzyme. In thecDNAs so obtained the N-terminal methionine was not included. However,as described in Example 13 genuine phosphatonin has an N-terminalmethionine present in its amino acid sequence. Therefore, in oneembodiment of the phosphatonin polypeptide of the invention the aminoacid sequence of the polypeptide includes the amino acid Met added tothe N-terminus.

[0089] In another embodiment, the polypeptide of the invention can bepart of a fusion protein. This embodiment will be discussed furtherbelow.

[0090] The present invention further provides a polynucleotide encodinga phosphatonin polypeptide as described herein. Such polynucleotide maybe a DNA such as a cDNA, or an RNA such as mRNA or any other form ofnucleic acid including synthetic or modified derivatives and may encodethe polypeptide in a continuous sequence or in a number of sequencesinterrupted by intervening sequences. In whichever form it is present,the polynucleotide is an isolated polynucleotide in that it is removedfrom its naturally-occurring state. This aspect of the invention isbased on the cloning of the gene for human phosphatonin. In a preferredembodiment, the polynucleotide comprises the nucleotide sequence of FIG.8 or the one depicted in SEQ ID NO: 26, optionally including one or moremutations or deletions which do not substantially affect the activity ofthe polypeptide encoded thereby. Such mutations include those arisingfrom the degeneracy of the genetic code, as well as those giving rise toany of the amino acid mutations or deletions discussed above.Accordingly, by the employment of techniques routine to those skilled inmolecular biology, it is possible to use the nucleotide sequence of FIG.8 or SEQ ID NO: 26 to generate suitable polynucleotide sequences whichencode polypeptides useful in the present invention. As mentioned hereinbefore, the present invention also encompasses phosphatoninpolynucleotides, wherein the nucleotide sequence comprises sequentialnucleotide deletions from either the C-terminus or the N-terminus suchas those described in more detail below.

[0091] Extending the Polynucleotide Sequence of the Invention

[0092] As discussed in Example 4 and 13, the phosphatonin polynucleotideobtained by the expression library was not full-length at the 5′-end.The polynucleotide sequences encoding the phosphatonin polypeptides canthus be extended utilizing partial nucleotide sequence and variousmethods known in the art to detect upstream sequences such as promotersand regulatory elements; see also Example 13. Gobinda, (PCR MethodsApplic. 2 (1993), 318-322) discloses “restriction-site” polymerase chainreaction (PCR) as a direct method which uses universal primers toretrieve unknown sequence adjacent to a known locus. First, genomic DNAis amplified in the presence of primer to a linker sequence and a primerspecific to the known region. The amplified sequences are subjected to asecond round of PCR with the same linker primer and another specificprimer internal to the first one. Products of each round of PCR aretranscribed with an appropriate RNA polymerase and sequenced usingreverse transcriptase.

[0093] Inverse PCR can be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, Nucleic Acids Res.16 (1988), 8186). The primers may be designed using OLIGO(r) 4.06 PrimerAnalysis Software (1992; National Biosciences Inc, Plymouth Minn.), oranother appropriate program to be preferably 22-30 nucleotides inlength, to have a GC content of preferably 50% or more, and to anneal tothe target sequence at temperatures preferably about 68-72 C. The methoduses several restriction enzymes to generate a suitable fragment in theknown region of a gene. The fragment is then circularized byintramolecular ligation and used as a PCR template.

[0094] Capture PCR (Lagerstrom, PCR Methods Applic. 1 (1991), 111-119)is a method for PCR amplification of DNA fragments adjacent to a knownsequence in, e.g., human yeast artificial chromosome DNA. Capture PCRalso requires multiple restriction enzyme digestions and ligations toplace an engineered double-stranded sequence into an unknown portion ofthe DNA molecule before PCR.

[0095] Another method which may be used to retrieve unknown sequences isthat of Parker, (Nucleic Acids Res. 19 (1991), 3055-3060). Additionally,one can use PCR, nested primers and PromoterFinder libraries to walk ingenomic DNA (PromoterFinder™ Clontech (Palo Alto Calif.). This processavoids the need to screen libraries and is useful in finding intron/exonjunctions. Preferred libraries for screening for full length cDNAs areones that have been size-selected to include larger cDNAs. Also, randomprimed libraries are preferred in that they will contain more sequenceswhich contain the 5′ and upstream regions of genes. A randomly primedlibrary may be particularly useful if an oligo d(T) library does notyield a full-length cDNA. Furthermore, direct sequencing of primerextension products may be employed. Genomic libraries are useful forextension into the 5′ nontranslated regulatory region. Capillaryelectrophoresis may be used to analyze the size or confirm thenucleotide sequence of sequencing or PCR products; see, e.g., Sambrook,supra. Systems for rapid sequencing are available from Perkin Elmer,Beckmann Instruments (Fullerton Calif.), and other companies.

[0096] Computer-assisted Identification of Phosphatonin Polypeptides andtheir Encoding Genes

[0097] BLAST2, which stands for Basic Local Alignment Search Tool(Altschul, Nucleic Acids Res. 25 (1997), 3389-3402; Altschul, J. Mol.Evol. 36 (1993), 290-300;-Altschul, J. Mol. Biol. 215 (1990), 403-410),can be used to search for local sequence alignments. BLAST producesalignments of both nucleotide and amino acid sequences to determinesequence similarity. Because of the local nature of the alignments,BLAST is especially useful in determining exact matches or inidentifying homologs. The fundamental unit of BLAST algorithm output isthe High-scoring Segment Pair (HSP). An HSP consists of two sequencefragments of arbitrary but equal lengths whose alignment is locallymaximal and for which the alignment score meets or exceeds a thresholdor cutoff score set by the user. The BLAST approach is to look for HSPsbetween a query sequence and a database sequence, to evaluate thestatistical significance of any matches found, and to report only thosematches which satisfy the user-selected threshold of significance. Theparameter E establishes the statistically significant threshold forreporting database sequence matches. E is interpreted as the upper boundof the expected frequency of chance occurrence of an HSP (or set ofHSPs) within the context of the entire database search. Any databasesequence whose match satisfies E is reported in the program output.

[0098] Analogous computer techniques using BLAST (Altschul, 1997, 1993and 1990, supra) are used to search for identical or related moleculesin nucleotide databases such as GenBank or EMBL. This analysis is muchfaster than multiple membrane-based hybridizations. In addition, thesensitivity of the computer search can be modified to determine whetherany particular match is categorized as exact or homologous. The basis ofthe search is the product score which is defined as:

% sequence identity×% maximum BLAST score

100

[0099] and it takes into account both the degree of similarity betweentwo sequences and the length of the sequence match. For example, with aproduct score of 40, the match will be exact within a 1-2% error; and at70, the match will be exact. Homologous molecules are usually identifiedby selecting those which show product scores between 15 and 40, althoughlower scores may identify related molecules.

[0100] Examples of the different possible applications of thephosphatonin polynucleotides and polypeptides according to the inventionas well as molecules derived from them will be described in detail inthe following.

[0101] Phosphatonin Polynucleotides and Polypeptides

[0102] The phosphatonin was isolated from a cDNA library constructedfrom MRNA extracted from a meningeal phosphaturic-mesenchymal-tumourresected from a patient suffering from oncogenic hypophosphatemicosteomalacia; see Example 4 and 13.

[0103] The phosphatonin nucleotide sequence identified as SEQ ID NO: 1was assembled from partially homologous (“overlapping”) sequencesobtained from related DNA clones. The overlapping sequences wereassembled into a single contiguous sequence of high redundancy (usuallythree to five overlapping sequences at each nucleotide position),resulting in a final sequence identified as SEQ ID NO: 1. SEQ ID NO: 26containing the additional nucleotide sequence encoding the 5′-terminusof phosphatonin has been isolated from the same tumor cDNA libraryconstructed for the isolation of the clones resulting in SEQ ID NO: 1.Therefore, SEQ ID NO: 1 and 26 and the translated SEQ ID NO:2 and 27,respectively, are sufficiently accurate and otherwise suitable for avariety of uses well known in the art and described further below. Forinstance, SEQ ID NO: 1 and 26 are useful for designing nucleic acidhybridization probes that will detect nucleic acid sequences containedin SEQ ID NO: 1 and 26. These probes will also hybridize to nucleic acidmolecules in biological samples, thereby enabling a variety of forensicand diagnostic methods of the invention. Similarly, polypeptidesidentified from SEQ ID NO:2 or 27 may be used to generate antibodieswhich bind specifically to phosphatonin.

[0104] Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidescause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

[0105] Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO: 1 and 26 and the predicted translated amino acid sequence identifiedas SEQ ID NO:2 and 27, respectively, but also means for the cloning ofthe cDNA and genomic DNA corresponding to the nucleotide sequence in SEQID NO:1 and 26. The nucleotide sequence of the so obtained phosphatoninclones can readily be determined by sequencing the clone in accordancewith known methods. The predicted phosphatonin amino acid sequence canthen be verified from such cDNA or genomic clones. Moreover, the aminoacid sequence of the protein encoded by the obtained clones can also bedirectly determined by peptide sequencing or by expressing the proteinin a suitable host cell, collecting the protein, and determining itssequence and function according to the methods described herein.

[0106] The present invention also relates to the phosphatonin genecorresponding to SEQ ID NO: 1 and 26. The phosphatonin gene can beisolated in accordance with known methods using the sequence informationdisclosed herein. Such methods include preparing probes or primers fromthe disclosed sequence and identifying or amplifying the phosphatoningene from appropriate sources of genomic material.

[0107] Also provided in the present invention are species homologs ofphosphatonin. Species homologs may be isolated and identified by makingsuitable probes or primers from the sequences provided herein andscreening a suitable nucleic acid source for the desired homologue.

[0108] Thus, by the provision of the nucleotide sequence of SEQ ID NO: 1and 26 as well as those encoding the amino acid sequence depicted in SEQID NO: 2 and 27, it is possible to isolate identical or similar nucleicacid molecules which encode phosphatonin proteins from other species ororganisms, in particular orthologous phosphatonin genes from mammalsother than human. The term “orthologous” as used herein means homologoussequences in different species that arose from a common ancestor geneduring speciation. Orthologous genes may or may not be responsible for asimilar function; see, e.g., the glossary of the “Trends Guide toBioinformatics”, Trends Supplement 1998, Elsevier Science.

[0109] The phosphatonin polypeptides can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

[0110] Phosphatonin polypeptides are preferably provided in an isolatedform, and preferably are substantially purified. A recombinantlyproduced version of a phosphatonin polypeptide, including the secretedpolypeptide, can be substantially purified by the one-step methoddescribed in Smith and Johnson, Gene 67 (1988), 31-40. Phosphatoninpolypeptides also can be purified from natural or recombinant sourcesusing antibodies of the invention raised against the phosphatoninprotein in methods which are well known in the art.

[0111] Polynucleotide and Polypeptide Variants

[0112] “Variant” refers to a polynucleotide or polypeptide differingfrom the phosphatonin polynucleotide or polypeptide, but retainingessential properties thereof such as the immunological and preferablybiological activity referred to above. Generally, variants are overallclosely similar, and, in many regions, identical to the phosphatoninpolynucleotide or polypeptide.

[0113] Such polynucleotides comprise those which encode fragments,analogues or derivatives and in particular orthologues of theabove-described phosphatonin proteins and differ, for example, by way ofamino acid and/or nucleotide deletion(s), insertion(s), substitution(s),addition(s) and/or recombination(s) or any other modification(s) knownin the art either alone or in combination from the above-described aminoacid sequences or their uriderlying nucleotide sequence(s). Methods forintroducing such modifications in the nucleic acid molecules accordingto the invention are well-known to the person skilled in the art. Allsuch fragments, analogues and derivatives of the protein of theinvention are included within the scope of the present invention, aslong as the essential characteristic immunological and/or biologicalproperties as defined above remain unaffected in kind.

[0114] The term “variant” means in this context that the nucleotide andtheir encoded amino acid sequence, respectively, of thesepolynucleotides differs from the sequences of the above-describedphosphatonin polynucleotides and polypeptides in one or more nucleotidepositions and are highly homologous to said nucleic acid molecules.Homology is understood to refer to a sequence identity of at least 40%,preferably 50%, more preferably 60%, still more preferably 70%,particularly an identity of at least 80%, preferably more than 90% andstill more preferably more than 95%. The deviations from the sequencesof the nucleic acid molecules described above can, for example, be theresult of nucleotide substitution(s), deletion(s), addition(s),insertion(s) and/or recombination(s); see supra. Homology can furtherimply that the respective nucleic acid molecules or encoded proteins arefunctionally and/or structurally equivalent. The nucleic acid moleculesthat are homologous to the nucleic acid molecules described above andthat are derivatives of said nucleic acid molecules are, for example,variations of said nucleic acid molecules which represent modificationshaving the same biological function, in particular encoding proteinswith the same or substantially the same biological function. They may benaturally occurring variations, such as sequences from other mammals, ormutations. These mutations may occur naturally or may be obtained bymutagenesis techniques. The allelic variations may be naturallyoccurring allelic variants as well as synthetically produced orgenetically engineered variants; see supra.

[0115] By a polynucleotide having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence of thepresent invention, it is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding thephosphatonin polypeptide. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. The query sequencemay be an entire sequence shown of SEQ ID NO: 1 or 26, the ORF (openreading frame), or any fragment specified as described herein.

[0116] As a practical matter, whether any particular nucleic acidmolecule or polypeptide is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98% or 99% identical to a nucleotide sequence of the presenceinvention can be determined conventionally using known computerprograms. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag et al. (Comp. App. Biosci. 6 (1990), 237-245.) In a sequencealignment the query and subject sequences are both DNA sequences. An RNAsequence can be compared by converting U's to T's. The result of saidglobal sequence alignment is in percent identity. Preferred parametersused in a FASTDB alignment of DNA sequences to calculate percentidentify are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, JoiningPenalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5,Gap Size Penalty 0.05, Window Size=500 or the length of the subjectnucleotide sequence, whichever is shorter.

[0117] If the subject sequence is shorter than the query sequencebecause of 5′ or 3′ deletions, not because of internal deletions, amanual correction must be made to the results. This is because theFASTDB program does not account for 5′ and 3′ truncations of the subjectsequence when calculating percent identity. For subject sequencestruncated at the 5′ or 3′ ends, relative to the query sequence, thepercent identity is corrected by calculating the number of bases of thequery sequence that are 5′ and 3′ of the subject sequence, which are notmatched/aligned, as a percent of the total bases of the query sequence.Whether a nucleotide is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thiscorrected score is what is used for the purposes of the presentinvention. Only bases outside the 5′ and 3′ bases of the subjectsequence, as displayed by the FASTDB alignment, which are notmatched/aligned with the query sequence, are calculated for the purposesof manually adjusting the percent identity score.

[0118] For example, a 90 base subject sequence is aligned to a 100 basequery sequence to determine percent identity. The deletions occur at the5′ end of the subject sequence and therefore, the FASTDB alignment doesnot show a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

[0119] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a query amino acid sequence of the presentinvention, it is intended that the amino acid sequence of the subjectpolypeptide is identical to the query sequence except that the subjectpolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the query amino acid sequence. In other words,to obtain a polypeptide having an amino acid sequence at least 95%identical to a query amino acid sequence, up to 5% of the amino, acidresidues in the subject sequence may be inserted, deleted, added orsubstituted with another amino acid. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0120] As a practical matter, whether any particular polypeptide is atleast 40%, 50%, 60%, 70%, 80%; 90%, 95%, 96%, 97%, 98% or 99% identicalto, for instance, the amino acid sequences shown in SEQ ID NO: 2 or 27can be determined conventionally using known computer programs. Apreferred method for determining the best overall match between a querysequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, can be determined usingthe FASTDB computer program based on the algorithm of Brutlag et al.(Comp. App. Biosci. 6 (1990), 237-245). In a sequence alignment thequery and subject sequences are either both nucleotide sequences or bothamino acid sequences. The result of said global sequence alignment is inpercent identity. Preferred parameters used in a FASTDB amino acidalignment are: Matrix=PAMO, k-tuple=2, Mismatch Penalty=1, JoiningPenalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter.

[0121] If the subject sequence is shorter than the query sequence due toN- or C-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for ihe purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

[0122] For example, a 90 amino acid residue subject sequence is alignedwith a 100 residue query sequence to determine percent identity. Thedeletion occurs at the N-terminus of the subject sequence and therefore,the FASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

[0123] The phosphatonin variants may contain alterations in the codingregions, non-coding regions, or both. Especially preferred arepolynucleotide variants containing alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. Nucleotide variants producedby silent substitutions due to the degeneracy of the genetic code arepreferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids aresubstituted, deleted, or added in any combination are also preferred.Phosphatonin polynucleotide variants can be produced for a variety ofreasons, e.g., to optimize codon expression for a particular host(change codons in the human mRNA to those preferred by a bacterial hostsuch as E. coli).

[0124] Naturally occurring phosphatonin variants are called “allelicvariants,” and refer to one of several alternate forms of a geneoccupying a given locus on a chromosome of an organism. (Genes II,Lewin, B., ed., John Wiley & Sons, New York (1985) and updatedversions). These allelic variants can vary at either the polynucleotideand/or polypeptide level. Alternatively, non-naturally occurringvariants may be produced by mutagenesis techniques or by directsynthesis.

[0125] Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the phosphatonin polypeptides. For instance, one ormore amino acids can be deleted from the N-terminus or C-terminus of theprotein without substantial loss of biological function. The authors ofRon, J. Biol. Chem. 268 (1993), 2984-2988, reported variant KGF proteinshaving heparin binding activity even after deleting 3, 8, or 27amino-terminal amino acid residues. Similarly, Interferon gammaexhibited up to ten times higher activity after deleting 8-10 amino acidresidues from the carboxy terminus of this protein. (Dobeli, J.Biotechnology 7 (1988), 199-216).

[0126] Moreover, ample evidence demonstrates that variants often retaina biological activity similar to that of the naturally occurringprotein. For example, Gayle and coworkers (J. Biol. Chem. 268 (1993);22105-22111) conducted extensive mutational analysis of human cytokineIL-1a. They used random mutagenesis to generate over 3,500 individualIL-1a mutants that averaged 2.5 amino acid changes per variant over theentire length of the molecule. Multiple mutations were examined at everypossible amino acid position. The investigators found that “[m]ost ofthe molecule could be altered with little effect on either [binding orbiological activity]”; see Abstract. In fact, only 23 unique amino acidsequences, out of more than 3,500 nucleotide sequences examined,produced a protein that significantly differed in activity fromwild-type.

[0127] Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the protein will likelybe retained when less than the majority of the residues of the proteinare removed from the N-terminus or C-terminus. Whether a particularpolypeptide lacking N- or C-terminal residues of a protein retains suchimmunogenic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art. Furthermore, using thePESTFIND program (Rogers, Science 234 (1986), 364-368), PEST sequences(rich in proline, glutamic acid, serine, and threonine) can beidentified, which are characteristically present in unstable proteins.Such sequences may be removed from the phosphatonin proteins in order toincrease the stability and optionally the activity of the proteins.Methods for introducing such modifications in the nucleic acid moleculesaccording to the invention are well-known to the person skilled in theart.

[0128] Thus, the invention further includes phosphatonin polypeptidevariants which show substantial biological activity. Such variantsinclude deletions, insertions, inversions, repeats, and substitutionsselected according to general rules known in the art so as have littleeffect on activity. For example, guidance concerning how to makephenotypically silent amino acid substitutions is provided in Bowie,Science 247 (1990), 1306-1310, wherein the authors indicate that thereare two main strategies for studying the tolerance of an amino acidsequence to change.

[0129] The first strategy exploits the tolerance of amino acidsubstitutions by natural selection during the process of evolution. Bycomparing amino acid sequences in different species, conserved aminoacids can be identified. These conserved amino acids are likelyimportant for protein function. In contrast, the amino acid positionswhere substitutions have been tolerated by natural selection indicatesthat these positions are not critical for protein function. Thus,positions tolerating amino acid substitution could be modified whilestill maintaining biological activity of the protein.

[0130] The second strategy uses genetic engineering to introduce aminoacid changes at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244 (1989), 1081-1085) The resulting mutant molecules can thenbe tested for biological activity.

[0131] As the authors state, these two strategies have revealed thatproteins are surprisingly tolerant of amino acid substitutions. Theauthors further indicate which amino acid changes are likely to bepermissive at certain amino acid positions in the protein. For example,most buried (within the tertiary structure of the protein) amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Moreover, tolerated conservativeamino acid substitutions involve replacement of the aliphatic orhydrophobic amino acids Ala, Val, Leu and Ile; replacement of thehydroxyl residues Ser and Thr; replacement of the acidic residues Aspand Glu; replacement of the amide residues Asn and Gln, replacement ofthe basic residues Lys, Arg, and His; replacement of the aromaticresidues Phe, Tyr, and Trp, and replacement of the small-sized aminoacids Ala, Ser, Thr, Met, and Gly.

[0132] Besides conservative amino acid substitution, variants ofphosphatonin include (i) substitutions with one or more of thenon-conserved amino acid residues, where the substituted amino acidresidues may or may not be one encoded by the genetic code, or (ii)substitution with one or more of amino acid residues having asubstituent group, or (iii) fusion of the mature polypeptide withanother compound, such as a compound to increase the stability and/orsolubility of the polypeptide (for example, polyethylene glycol), or(iv) fusion of the polypeptide with additional amino acids, such as anIgG Fc fusion region peptide, or leader or secretary sequence, or asequence facilitating purification. Such variant polypeptides are deemedto be within the scope of those skilled in the art from the teachingsherein.

[0133] For example, phosphatonin polypeptide variants containing aminoacid substitutions of charged amino acids with other charged or neutralamino acids may produce proteins with improved characteristics, such asless aggregation. Aggregation of pharmaceutical formulations bothreduces activity and increases clearance due to the aggregate'simmunogenic activity; see, e.g. Pinckard, Clin. Exp. Immunol. 2 (1967),331-340; Robbins, Diabetes 36 (1987), 838-845; Cleland, Crit. Rev.Therapeutic Drug Carrier Systems 10 (1993), 307-377.

[0134] Polynucleotide and Polypeptide Fragments

[0135] In the present invention, a “polynucleotide fragment” refers to ashort polynucleotide having a nucleic acid sequence contained in SEQ IDNO: 1 or 26. The short nucleotide fragments are preferably at leastabout 15 nt, and more preferably at least about 20 nt, still morepreferably at least about 30 nt, and even more preferably, at leastabout 40 nt in length. A fragment “at least 20 nt in length,” forexample, is intended to include 20 or more contiguous bases from thecDNA sequence contained in the nucleotide sequence shown in SEQ ID NO: 1or 26. These nucleotide fragments are useful as diagnostic probes andprimers as discussed herein. Of course, larger fragments (e.g., 50, 150,500, 600, 1000 nucleotides) are preferred.

[0136] Moreover, representative examples of phosphatonin polynucleotidefragments include, for example, fragments having a sequence from aboutnucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300,301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750,751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100,1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400,1401-1450,1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700,1701-1750, 1751-1800, 1801-1850, 1851-1900, or 1901-1950 of SEQ ID NO:26. In this context “about” includes the particularly recited ranges,larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus or at both termini. Preferably, these fragments encode apolypeptide which has biological activity. More preferably, thesepolynucleotides can be used as probes or primers as discussed herein.

[0137] In the present invention, a “polypeptide fragment” refers to ashort amino acid sequence contained in SEQ ID NO: 2 or 27. Proteinfragments may be “free-standing”, or comprised within a largerpolypeptide of which the fragment forms a part or region, mostpreferably as a single continuous region. Representative examples ofpolypeptide fragments of the invention, include, for example, fragmentsfrom about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120,121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280,281-300, 301-320, or 321-340, 341-360, 361-380, 381-400, 401-420,421-440, 441-460, 461-480, 481-500, and 501-520 to the end of the codingregion in SEQ ID NO: 26. Moreover, polypeptide fragments can be about20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 aminoacids in length. In this context “about” includes the particularlyrecited ranges, larger or smaller by several (5, 4, 3, 2, or 1) aminoacids, at either extreme or at both extremes.

[0138] Preferred polypeptide fragments include the phosphatonin proteinhaving a continuous series of deleted residues from the amino or thecarboxy terminus, or both. For example, any number of amino acids,ranging from 1-60, can be deleted from the amino terminus of thephosphatonin polypeptide. Similarly, any number of amino acids, rangingfrom 1-30, can be deleted from the carboxy terminus of the phosphatoninprotein. Furthermore, any combination of the above amino and carboxyterminus deletions are preferred. Similarly, polynucleotide fragmentsencoding these phosphatonin polypeptide fragments are also preferred.Particularly, N-terminal deletions of the phosphatonin polypeptide canbe described by the general formula m-525, where m is an integer from 2to 520 where m corresponds to the position of the amino acid residueidentified in SEQ ID NO: 27.

[0139] Also preferred are phosphatonin polypeptide and polynucleotidefragments characterized by structural or functional domains. Preferredembodiments of the invention include fragments that comprise alpha-helixand alpha-helix forming regions (“alpha-regions”), beta-sheet andbeta-sheet-forming regions (“beta-regions”), turn and turn-formingregions (“tum-regions”), coil and coil-forming regions (“coil-regions”),hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forrning regions,substrate binding region, and high antigenic index regions. As set outin the Figures, such preferred regions include Garnier-Robson-alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasmanalpha-regions, beta-regions, and turn-regions, Kyte-Doolittlehydrophilic regions and hydrophobic regions, Eisenberg alpha and betaamphipathic regions, Karplus-Schulz flexible regions, Eminisurface-forming regions, and Jameson-Wolf high antigenic index regions.Polyp eptide fragments of SEQ ID NO: 2 and 27 falling within conserveddomains are specifically contemplated by the present invention and shownin the Figures. Moreover, polynucleotide fragments encoding thesedomains are also contemplated.

[0140] Other preferred fragments are biologically active phosphatoninfragments. Biologically active fragments are those exhibiting activitysimilar, but not necessarily identical, to an activity of thephosphatonin polypeptide. The biological activity of the fragments mayinclude an improved desired activity, or a decreased undesirableactivity.

[0141] However, many polynucleotide sequences, such as EST sequences,are publicly available and are accessible through sequence databases.Some of these sequences may be related to SEQ ID NO: 1 or 26 and mayhave been publicly available prior to conception of the presentinvention. Preferably, such related polynucleotides are specificallyexcluded from the scope of the present invention. To list every relatedsequence would be cumbersome.

[0142] Accordingly, preferably excluded from the present invention areone or more polynucleotides comprising a nucleotide sequence describedby the general formula of a-b, where a is any integer between 1 to 1989of SEQ ID NO: 26, b is an integer of 15 to 1989, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:26, and where the b is greater than or equal to a +14.

[0143] Epitopes & Antibodies

[0144] In the present invention, “epitopes” refer to phosphatoninpolypeptide fragments having antigenic or immunogenic activity in ananimal, e.g., a rat, a rabbit, a human, a mouse (including a transgenicmouse which carry human immunoglobulin genes and produce human antibodymolecules), and so on. A preferred embodiment of the present inventionrelates to a phosphatonin polypeptide fragment comprising an epitope, aswell as the polynucleotide encoding this fragment. A region of a proteinmolecule to which an antibody can bind is defined as an “antigenicepitope.” In contrast, an “immunogenic epitope” is defined as a part ofa protein that elicits an antibody response; see, for instance, Geysen,Proc. Natl. Acad. Sci. USA 81 (1983); 3998-4002. Fragments whichfunction as epitopes may be produced by any conventional means; see,e.g., Houghten, Proc. Natl. Acad. Sci. USA 82 (1985), 5131-5135 furtherdescribed in U.S. Pat. No. 4,631,211.

[0145] In the present invention, antigenic epitopes preferably contain asequence of at least seven, more preferably at least nine, and mostpreferably between about 15 to about 30 amino acids. Antigenic epitopesare useful to raise antibodies, including monoclonal antibodies, thatspecifically bind the epitope; see, for instance, Wilson, Cell 37(1984), 767-778; Sutcliffe, Science 219 (1983), 660-666.)

[0146] Similarly, immunogenic epitopes can be used to induce antibodiesaccording to methods well known in the art; see, for instance,Sutcliffe, supra; Wilson, supra; Chow, Proc. Natl. Acad. Sci. USA 82(1985), 910-914; and Bittle, J. Gen. Virol. 66 (1985); 2347-2354. Apreferred immunogenic epitope includes the soluble protein. Theimmunogenic epitopes may be presented together with a carrier protein,such as an albumin, to an animal system (such as rabbit or mouse) or, ifit is long enough (at least about 25 amino acids), without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting.)

[0147] Using the computer program GCG-Peptide-structure (Rice, ProgrammeManual for the EGCG package, Cambridge, CB10 1RQ England: Hinxton Hall;1995) available from the Human Genome Resource Centre(http://www.hgmp.mrc.ac.uk/homepage.html), SEQ ID NO:2 was foundantigenic at amino acids regions shown in FIG. 4. Thus, these regionscould be used as epitopes to produce antibodies against the proteinencoded by SEQ ID NO: 1. Preferably, the antibody of the presentinvention specifically recognizes an epitope combined in or formed withamino acid residues 1 to 96, more preferably residues 47 to 96 of SEQ IDNO: 27.

[0148] As used herein, the term “antibody” (Ab) or “monoclonal antibody”(Mab) is meant to include intact molecules as well as antibody fragments(such as, for example, Fab and F(ab′)2 fragments) which are capable ofspecifically binding to protein. Fab and F(ab′)2 fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding than an intact antibody;see, e.g., Wahl, J. Nucl. Med. 24 (1983), 316-325. Thus, these fragmentsare preferred, as well as the products of a FAB or other immunoglobulinexpression library. Moreover, antibodies of the present inventioninclude chimeric, single chain, humanized antibodies, human antibodiesobtainable by or from phage display, a transgenic mouse carrying humanimmunoglobulin genes and/or human chromosomes, isolated immune cellsfrom human body, in vitro or ex vivo immunization of human immune cells,or any other available methods.

[0149] In another embodiment, the present invention relates to a nucleicacid molecule which hybridizes with the complementary strand of thephosphatonin polynucleotide of the invention and which encodes a mutatedversion of the protein as defined above which has lost itsimmunological, preferably one of its biological activities. Thisembodiment may prove useful for, e.g., generating dominant mutantalleles of the above-described phosphatonin proteins. Said mutatedversion is preferably generated by substitution, deletion and/oraddition of 1 to 5 or 5 to 10 amino acid residues in the amino acidsequence of the above-described wild type proteins. For example, any oneof the putative functional and structural motifs shown in FIG. 15 can bemutated and altered, substituted or otherwise modified, either alone orin combination.

[0150] Vectors, Host Cells and Protein Production

[0151] The present invention also relates to vectors containing thephosphatonin polynucleotide, host cells, and the production ofpolypeptides by recombinant techniques. The vector may be, for example,a phage, plasmid, viral, or retroviral vector. Retroviral vectors may bereplication competent or replication defective. In the latter case,viral propagation generally will occur only in complementing host cells.

[0152] Phosphatonin polynucleotides may be joined to a vector containinga selectable marker for propagation in a host. Generally, a plasmidvector is introduced in a precipitate, such as a calcium phosphateprecipitate, or in a complex with a charged lipid. If the vector is avirus, it may be packaged in vitro using an appropriate packaging cellline and then transduced into host cells.

[0153] The phosphatonin polynucleotide insert should be operativelylinked to an appropriate promoter, such as the phage lambda PL promoter,the E. coli lac, trp, phoA and tac promoters, the SV40 early and latepromoters and promoters of retroviral LTRs, to name a few. Othersuitable promoters will be known to the skilled artisan. The expressionconstructs will further contain sites for transcription initiation,termination, and, in the transcribed region, a ribosome binding site fortranslation. The coding portion of the transcripts expressed by theconstructs will preferably include a translation initiating codon at thebeginning and a termination codon (UAA, UGA or UAG) appropriatelypositioned at the end of the polypeptide to be translated.

[0154] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase, G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; andplant cells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art. Among vectors preferredfor use in bacteria include pQE70, pQE60 and pQE-9, available fromQIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a,pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; andptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from PharmaciaBiotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXTI and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia. Other suitable vectors will be readilyapparent to the skilled artisan.

[0155] Furthermore, one could use, e.g., a mammalian cell that alreadycomprises in its genome a nucleic acid molecule encoding a phosphatoninpolypeptide as described above, but does not express the same or not inan appropriate manner due to, e.g., a weak promoter, and introduce intothe mammalian cell an expression control sequence such as a strongpromoter in close proximity to the endogenous nucleic acid moleculeencoding said phosphatonin polypeptide so as to induce expression of thesame.

[0156] In this context the term “expression control sequence” denotes anucleic acid molecule that can be used to increase the expression of thephosphatonin polypeptide, due to its integration into the genome of acell in close proximity to the phosphatonin encoding gene. Suchregulatory sequences comprise promoters, enhancers, inactivated silencerintron sequences, 3UTR and/or 5′UTR coding regions, protein and/or RNAstabilizing elements, nucleic acid molecules encoding a regulatoryprotein, e.g., a transcription factor, capable of inducing or triggeringthe expression of the phosphatonin gene or other gene expression controlelements which are known to activate gene expression and/or increase theamount of the gene product. The introduction of said expression controlsequence leads to increase and/or induction of expression ofphosphatonin polypeptides, resulting in the end in an increased amountof phosphatonin polypeptides in the cell. Thus, the present invention isaiming at providing de novo and/or increased expression of phosphatoninpolypeptides.

[0157] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis, Basic Methods In Molecular Biology(1986). It is specifically contemplated that phosphatonin polypeptidesmay in fact be expressed by a host cell lacking a recombinant vector.

[0158] Phosphatonin polypeptides can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

[0159] Phosphatonin polypeptides can also be recovered from productspurified from natural sources, including bodily fluids, tissues andcells, whether directly isolated or cultured; products of chemicalsynthetic procedures; and products produced by recombinant techniquesfrom a prokaryotic or eukaryotic host, including, for example,bacterial, yeast, higher plant, insect, and mammalian cells. Dependingupon the host employed in a recombinant production procedure, thephosphatonin polypeptides may be glycosylated or may benon-glycosylated. In addition, phosphatonin polypeptides may alsoinclude an initial (modified) methionine residue, in some cases as aresult of host-mediated processes. Thus, it is well known in the artthat the N-terminal methionine encoded by the translation initiationcodon generally is removed with high efficiency from any protein aftertranslation in all eukaryotic cells. While the N-terminal methionine onmost proteins also is efficiently removed in most prokaryotes, for someproteins, this prokaryotic removal process is inefficient, depending onthe nature of the amino acid to which the N-terminal methionine iscovalently linked.

[0160] In a particularly preferred embodiment, the present inventionrelates to a process for isolating a phosphatonin polypeptide comprisingthe steps of:

[0161] (a) culturing tumor-conditioned media or osteosarcoma cells toconfluence in serum supplemented media (DMEM Eagles/10%FCS/glutamine/antimycotic (DMFCS);

[0162] (b) incubating the cells on alternate days in serum free mediaDMEM Eagles/glutamine/antimycotic antibiotic (DM) up to five hours;

[0163] (c) collecting conditioned serum free media from the cells andequilibrating the conditioned media to 0.06M sodium phosphate pH 7.2 and0.5 M NaCl (PBS);

[0164] (d) subjecting the media from (c) to an equilibrated column ofconcanavilin A sepharose;

[0165] (e) washing the column extensively with PBS;

[0166] (f) eluting the concanavalin A column with PBS supplemented with0.5 M (-methyl-D-glucopyranoside;

[0167] (g) subjecting the eluted material from (f) to cation exchangechromatography; and

[0168] (h) eluting phosphatonin polypeptide containing fractions with0.5 M NaCl.

[0169] The above-described method is illustrated in Example 1.

[0170] Another subject of the invention is a method for the preparationof phosphatonin polypeptides which comprises the cultivation of hostcells according to the invention which, due to the presence of a vectoror a polynucleotide according to the invention or an exogenousexpression control sequence, are able to express such a polypeptide,under conditions which allow expression of the polypeptide andrecovering of the so-produced polypeptide from the culture. It is alsoto be understood that the proteins can be expressed in a cell freesystem using for example in vitro translation assays known in the art.

[0171] Hence, in a still further embodiment, the present inventionrelates to a phosphatonin polypeptide or an immunologically and/orbiologically active fragment thereof encoded by the polynucleotide ofthe invention or produced by a method of as described above. Likewisephosphatonin polypeptides are within the scope of the present inventionwhich are obtainable by proteolytic cleavage of the above describedphosphatonin polypeptides by a PHEX metallopeptidase.

[0172] It will be apparent to those skilled in the art that the proteinof the invention can be further coupled to other moieties as describedabove for, e.g., drug targeting and imaging applications. Such couplingmay be conducted chemically after expression of the protein to site ofattachment or the coupling product may be engineered into the protein ofthe invention at the DNA level. The DNAs are then expressed in asuitable host system, and the expressed proteins are collected andrenatured, if necessary.

[0173] As explained above, phosphatonin comprising the amino acidsequence depicted in SEQ ID. NO: 27 has an N-terminus with twocysteines, one of which is optimally placed for the formation of homo-and/or heterodimers. Thus, it is envisaged that within the cellfunctional phosphatonin is present as a homo- or heterodimer. Therefore,in a preferred embodiment of the present invention, phosphatonin forms ahomo- or heterodimer:

[0174] On the other hand, it could be shown in accordance with thepresent invention, that recombinant truncated phosphatonin that lacksboth cysteine residues in its amino acid sequence, hasphosphate-retaining properties thus useful in therapeutic applications.Accordingly, in another preferred embodiment of the present invention,the cysteine residues in the phosphatonin polypeptide of the presentinvention are substituted, deleted and/or blocked, preferably such thatthe phosphatonin polypeptide is no longer capable of forming homo-and/or heterodimers, for example, due to disulfide bridges.

[0175] Regulation of a Phosphate Metabolism

[0176] As mentioned hereinbefore, the phosphatonin polypeptide of thepresent invention is capable of regulating phosphate metabolism indifferent ways. Thus, in one embodiment, the present invention relatesto a phosphatonin polypeptide having phosphatonin activity in that ithas at least one of the following activities:

[0177] (a) it is capable of down-regulating sodium dependent phosphateco-transport;

[0178] (b) it is capable of up-regulating renal 25-hydroxy vitaminD3-24-hydroxylase; and/or

[0179] (c) it is capable of down-regulating renal25-hydroxy-D-1-(-hydroxylase.

[0180] In another embodiment, the present invention relates to aphosphatonin polypeptide having anti-phosphatonin activity in that ithas at least one of the following activities:

[0181] (a) it is capable of up-regulating sodium dependent phosphateco-transport;

[0182] (b) it is capable of down-regulating renal 25-hydroxy vitaminD3-24-hydroxylase; and/or

[0183] (c) it is capable of up-regulating renal25-hydroxy-D-1-(-hydroxylase.

[0184] In a particularly preferred embodiment of the present invention,the phosphatonin polypeptide comprises a bone mineral motif as describedabove and positively regulates bone mineralization.

[0185] In a still further embodiment, the present invention relates tophosphatonin polypeptides which have lost at least one of the abovedescribed activities. Such polypeptides may be mutant forms of thephosphatonin polypeptide of the present invention and can, e.g., be usedfor studying the effect of mutations in the phosphatonin encoding gene.In particular, such mutants may prove useful for the development ofdrugs that are capable of compensating a deficiency caused by the lossof one of the biological activities of the wildtype phosphatonin. Suchmutant forms of phosphatonin polypeptides may best be studied in thescreening methods described in more detail hereinbelow.

[0186] Phosphatonin Antibodies

[0187] Furthermore, as described above, the provision of thephosphatonin polypeptide of the present invention enables the productionof phosphatonin specific antibodies. In this respect, hybridomatechnology enables production of cell lines secreting antibody toessentially any desired substance that produces an immune response. RNAencoding the light and heavy chains of the immunoglobulin can then beobtained from the cytoplasm of the hybridoma. The 5′ end portion of theMRNA can be used to prepare cDNA to be inserted into an expressionvector. The DNA encoding the antibody or its immunoglobulin chains cansubsequently be expressed in cells, preferably mammalian cells.Depending on the host cell, renaturation techniques may be required toattain proper conformation of the antibody. If necessary, pointsubstitutions seeking to optimize binding may be made in the DNA usingconventional cassette mutagenesis or other protein engineeringmethodology such as is disclosed herein.

[0188] Thus, the present invention also relates to an antibodyspecifically recognizing the phosphatonin polypeptide of the invention.

[0189] In a preferred embodiment of the invention, said antibody is amonoclonal antibody, a polyclonal antibody, a single chain antibody,human or humanized antibody, primatized, chimerized or fragment thereofthat specifically binds said peptide or polypeptide also includingbispecific antibody, synthetic antibody, antibody fragment, such as Fab,Fv or scFv fragments etc., or a chemically modified derivative of any ofthese. The general methodology for producing antibodies is well-knownand has been described in, for example, K÷hler and Milstein, Nature 256(1975), 494 and reviewed in J. G. R. Hurrel, ed., “Monoclonal HybridomaAntibodies: Techniques and Applications”, CRC Press Inc., Boco Raron,Fla. (1982), as well as that taught by L. T. Mimms et al., Virology 176(1990), 604-619. Furthermore, antibodies or fragments thereof to theaforementioned peptides can be obtained by using methods which aredescribed, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”,CSH Press, Cold Spring Harbor, 1988.

[0190] For the production of antibodies in experimental animals, varioushosts including goats, rabbits, rats, mice, and others, may be immunizedby injection with polypeptides of the present invention or any fragmentor oligopeptide or derivative thereof which has immunogenic properties.Techniques for producing and processing polyclonal antibodies are knownin the art and are described in, among others, Mayer and Walker, eds.,“Immunochemical Methods in Cell and Molecular Biology”, Academic Press,London (1987). Polyclonal antibodies also may be obtained from ananimal, preferably a mammal. Methods for purifying antibodies are knownin the art and comprise, for example, immunoaffinity chromatography.Depending on the host species, various adjuvants or immunologicalcarriers may be used to increase immunological responses. Such adjuvantsinclude, but are not limited to, Freund's, complete or incompleteadjuvants, mineral gels such as aluminium hydroxide, and surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions and dinitrophenol. An example of a carrier, to which, forinstance, a peptide of the invention may be coupled, is keyhole limpethemocyanin (KLH).

[0191] The production of chimeric antibodies is described, for example,in WO89/09622. Methods for the production of humanized antibodies aredescribed in, e.g., EP-A1 0 239 400 and WO90/0786 1. A further source ofantibodies to be utilized in accordance with the present invention areso-called xenogenic antibodies. The general principle for the productionof xenogenic antibodies such as human antibodies in mice is describedin, e.g., WO 91/10741, WO 94/02602, WO 96/34096 and WO 96/33735.

[0192] In a preferred embodiment, the antibody of the invention has anaffinity of at least about 10-7 M, preferably at least about 10-8 M morepreferably at least about 10-9 M and most preferably at least about10-10 M. On the other hand, the phosphatonin antibody may have a bindingaffinity of about 105 M-1, preferably not higher than 107 M-1 ifstimulation of phosphatonin activity is envisaged and advantageously upto 1010 M-1 or more in case phosphatonin activity should be suppressed.As mentioned hereinbefore, the antibody of the present inventionpreferably specifically recognizes an epitope contained or formed withamino acid residues 1 to 46 and 47 to 525, and more preferably 1 to 46,and 47 to 96 of the amino acid sequence depicted in SEQ ID NO: 27.

[0193] Uses of the Phosphatonin Polynucleotides

[0194] The phosphatonin polynucleotides identified herein can be used innumerous ways as reagents. The, following description should beconsidered exemplary and utilizes known techniques.

[0195] There exists an ongoing need to identify new chromosome markers,since few chromosome marking reagents, based on actual sequence data(repeat polymorphisms), are presently available. Phosphatonin relatedpolynucleotides (genomic and/or CDNA) can be used to carry outrestriction analysis as described in detail (Rowe, Hum. Genet. 94:5(1994), 457-467; Benham, Genomics 12 (1992), 368-376; Gillett, Ann. Hum.Genet. 60(3) (1996), 201-211; Rowe, Nucleic Acids Res. 22(23) (1994),5135-5136). In particular, the use of microsatellites (Rowe, Hum. Genet.94:5 (1994), 457-467; Rowe, Nucleic Acids Res. 22(23) (1994), 5135-5136;Rowe, Hum. Genet. 93 (1994), 291-294; Rowe, Hum. Genet. 91 (1993),571-575; Rowe, Hum. Genet. 97 (1996), 345-352; Rowe, Hum. Genet. 89(1992), 539-542), and the isolation of informative markers usingirradiation-fusion-gene-transfer hybrids and ALU-PCR (Benham, Genomics12 (1992), 368-376) will enable the rapid isolation of highlyinformative methods for the screening of phosphatonin and derivativeinherited diseases. The above methodologies have been particularlysuccessful in the mapping and localization of the PHEX gene (MEPE isproposed to a PHEX substrate), and extensive mutation analysis hasrevealed structural regions and motifs prerequisite for PHEXbio-activity (Rowe, Hum. Mol. Genet. 6 (1997), 539-549; Rowe, Exp.Nephrol. 5 (1997), 355-363; Rowe, Current Opinion in Nephrology &Hypertension 7(4) (1998), 367-376; Rowe, Clinical and ExperimentalNephrology 2(3) (1998), 183-193), these same approaches can be used forphosphatonin. More recently powerful genome-wide linkage and screeningtechniques have been developed that rely on single nucleotidepolymorphisms (SNP's), and the use of a combination of gel-basedsequencing and high-density variation-detection DNA chips (Wang, Science280 (1998), 1077-1082). Recently SNP data has been made available on theinternet by the Center for Genome Research at the Whitehead Institutefor Biomedical Research in Cambridge, Mass., USA (Whitehead-MIT) athttp://www-genome.wi.mit.edu/SNP/human/index.html. This powerful newoligonucleotide-array based methodology will be the future route formolecular expression analysis, polymorphism and genotyping, and diseasemanagement (Wang, Science 280 (1998), 1077-1082; Chee, Science 274(1996), 610-614; Gentalen, Nucleic Acids Res. 27 (1999), 1485-1491;Hacia, Nucleic Acids Res. 26 (1998), 3865-3866; Lipshutz, Nat. Genet. 21(1999), 20-24; Fan, Eur. J. Hum. Genet. 6 (1998), 134). Given thesequence information for MEPE in this application the above newapproaches and technology will be used to address the areas described.The sequence may be mapped to a particular chromosome or to a specificregion of the chromosome using well known techniques. These include insitu hybridization to chromosomal spreads, flow-sorted chromosomalpreparations, or artificial chromosome constructions such as yeastartificial chromosomes, bacterial artificial chromosomes, bacterial P1constructions or single chromosome cDNA libraries as reviewed in Price(Blood Rev. 7 (1993), 127-134) and Trask (Trends Genet. 7 (1991),149-154). The technique of fluorescent in situ hybridization ofchromosome spreads has been described, among other places, in Verma,(1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York N.Y. Fluorescent in situ hybridization of chromosomalpreparations and other physical chromosome mapping techniques may becorrelated with additional genetic map data. Extensive mapping dataaccessible to the scientific community can be found on the internet atsites sponsored by the Human-Genome-Mapping-Project United Kingdom(HGMP-RC) http://www.hgmp.mrc.ac.uk/homepage.html, the NationalCollection of biological information (NCBI) sponsored by the NationalInstitute of Health USA (NIH), http://ww.ncbi.nlm.nih.gov/, also theCenter for Genome Research at the Whitehead Institute for BiomedicalResearch in Cambridge, Mass., USA (Whitehead-MIT)http://www-genome.wi.mit.edu/. Moreover, extensive microsatellite-mapsand related mapping tools covering the entire human genome can also beaccessed via Genethon (French Government sponsored database)http://www.genethon.fr/genethon_en.html. Seminal maps have also beenpublished in Science and Nature (see, for example, Dib, Nature 380(1996), 152-154), but for up to date data the internet sites should beconsulted. Correlation between the location of the gene encoding aphosphatonin polypeptide of the invention on a physical chromosomal mapand a specific feature, e.g., a hypo- or hyperphosphatemic disease mayhelp to delimit the region of DNA associated with this feature. Thenucleotide sequences of the subject invention may be used to detectdifferences in gene sequences between normal, carrier or affectedindividuals. Furthermore, the means and methods described herein can beused for marker-assisted animal breeding. The nucleotide sequence of thesubject invention may also be used to detect differences in thechromosomal location due to translocation, inversion, etc. among normal,carrier or affected individuals.

[0196] In the very least, the phosphatonin polynucleotides can be usedas molecular weight markers on Southern gels, as diagnostic probes forthe presence of a specific mRNA in a particular cell type, as a probe to“subtract-out” known sequences in the process of discovering novelpolynucleotides, for selecting and making oligomers for attachment to a“gene chip” or other support, to raise anti-DNA antibodies using DNAimmunization techniques, and as an antigen to elicit an immune response.In a preferred embodiment, the described phosphatonin oligonucleotidecomprises at least 15, preferably 20 nucleotides of nucleotides 1 to 335of SEQ ID NO: 26 or of corresponding nucleotide sequences degenerate tothis nucleotide sequence and/or capable of specifically hybridizing tothis nucleotide sequence. In the latter, it is preferred that thenucleotide sequence is at least 70% or more, preferably 80%, and mostpreferably 90% identical to the corresponding sequence in SEQ ID NO: 26or its degenerated nucleotide sequence.

[0197] Uses of Phosphatonin Polypeptides and Antibodies

[0198] Phosphatonin polypeptides and antibodies thereto can be used innumerous ways. The following description should be considered exemplaryand utilizes known techniques.

[0199] Phosphatonin polypeptides can be used to assay protein levels ina biological sample using antibody-based techniques. For example,protein expression in tissues can be studied with classicalimmunohistological methods; see, e.g., Jalkanen, J. Cell. Biol. 101(1985), 976-985; Jalkanen, J. Cell. Biol. 105 (1987), 3087-3096.) Otherantibody based methods useful for detecting protein gene expressioninclude immunoassays, such as the enzyme linked immunosorbent assay(ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labelsare known in the art and include enzyme labels, such as, glucoseoxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), andfluorescent labels, such as fluorescein and rhodamine, and biotin.

[0200] In addition to assaying protein levels in a biological sample,proteins can also be detected in vivo by imaging. Antibody labels ormarkers for in vivo imaging of protein include those detectable byX-radiography, NMR or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the antibody by labeling ofnutrients for the relevant hybridoma.

[0201] A protein-specific antibody or antibody fragment which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, 131I, 121In, 99mTc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously, orintraperitoneally) into the mammal. It will be understood in the artthat the size of the subject and the imaging system used will determinethe quantity of imaging moiety needed to produce diagnostic images. Inthe case of a radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries of 99mTc. The labeled antibody or antibody fragment willthen preferentially accumulate at the location of cells which containthe specific protein. In vivo tumor imaging is described in, e.g.,Burchiel, “Immunopharmacokinetics of Radiolabeled Antibodies and TheirFragrnents”, Chapter 13 in Tumor Imaging: The Radiochemical Detection ofCancer, Burchiel and Rhodes, eds., Masson Publishing Inc. (1982).

[0202] Thus, the invention provides a diagnostic method of a disorder,which involves (a) assaying the expression of phosphatonin polypeptidein cells or tissues, or the level of phosphatonin or its activefragments or epitopes in the body fluid of an individual; (b) comparingthe level of gene expression with a standard gene expression level,whereby an increase or decrease in the assayed phosphatonin polypeptidegene expression level compared to the standard expression level isindicative of a disorder.

[0203] Moreover, phosphatonin polypeptides can be used to treat disease.For example, patients can be administered phosphatonin polypeptides inan effort to increase or decrease serum phosphate level and/or improvethe impaired bone formation (X-Linked Hyophosphatemic Rickets, OncogenicHypophosphatemic Osteomalacia, Renal Failure, Osteoporosis, RenalOsteodystrophy, and so forth). It can activate or inhibit its receptorsto up- or down-regulate the expression of sodium dependent phosphateco-transporters. In addition, the phosphatonin gene promoter and/orenhancer element can be used in gene therapy applications for treatingphosphate metabolism-specific disorders, particularly X-LinkedHypophosphatemic Rickets. Also, possibly in skeletal-mineral lossdisorders where inappropriate gene regulation and/or post-translationalmodification of MEPE occurs due to undefined secondary or primarychanges (e.g., postmenopausal women, osteoposis, age related), wheresupplementation of the hormone (and/or agonists-antagonists to receptoror hormone) perhaps as an adjunct to hormone replacement therapy wouldrestore phosphate and bone-mineral balance. A key feature of MEPEbio-activity and, thus, disease-treatment is the prediction thatN-terminal sequence regulates renal phosphate uptake, and the C-terminus(notably regions associated with the MEPE-motif described earlier) ispre-requisite for normal bone mineralization and growth.

[0204] After renal-transplantation, chronic hyperphosphatemia or in somecases hypophosphatemia are key features that result in major clinicalcomplications. For example, renal transplantation of a normal kidneyinto a male HYP patient was reported to result in pathophysiologicalchanges in the normal transplanted kidney such that a “rickets-type”renal phosphate leak developed (Morgan, Arch. Intern. Med. 134 (1974),549-552). The clinical use of N-terminal-cleaved processed-fragments ofMEPE could result in effective anti-hypophosphatemic therapy. Incontrast, renal-transplantation cases that result in hyperphosphatemiacould be treated with whole recombinant MEPE or active derivativepeptides modeled on distinct N-terminal residues. Other diseases thatcould benefit from treatment with MEPE, MEPE derivative peptides,receptor antagonists-agonists (peptides could be modified to increasepotency and specificity of action) include renal osteodystrophy, renaltoxicity, Pagets disease of bone, autosomal-forms of rickets, certainforms of renal Fanconi syndrome. Moreover, if receptors are expressed ina range of tissues (intestines, etc.) as well as the kidney, then thepotential for treating patients with end stage renal disease exists(i.e. complete loss of kidney function).

[0205] Similarly, antibodies directed to phosphatonin polypeptides canalso be used to treat disease. For example, administration of anantibody directed to a phosphatonin polypeptide can bind and reduceoverproduction of the polypeptide. Similarly, administration of anantibody can activate the polypeptide, such as by binding to apolypeptide and cleaving it to a different activity form.

[0206] At the very least, the phosphatonin polypeptides can be used asmolecular weight markers on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods well known to those of skill in theart. Phosphatonin polypeptides can also be used to raise antibodies,which in turn are used to measure protein expression from a recombinantcell, as a way of assessing transformation of the host cell.

[0207] Furthermore, phosphatonin polynucleotides and polypeptides can beused in assays to test for one or more biological activities. Ifphosphatonin polynucleotides and polypeptides do exhibit activity in aparticular assay, it is likely that phosphatonin may be involved in thediseases associated with the biological activity. Therefore,phosphatonin could be used to treat the associated disease.

[0208] Regulatory Sequences of Phosphatonin Genes

[0209] In a further aspect the present invention relates to a regulatorysequence of a promoter naturally regulating the expression of apolynucleotide encoding the phosphatonin polypeptide of the inventiondescribed above or of a polynucleotide homologous to a polynucleotide ofthe invention. With methods well known in the art it is possible toisolate the regulatory sequences of the promoters that naturallyregulate the expression of the above-described DNA sequences. Forexample, using the above described nucleic acid molecules as probes agenomic library consisting of human genomic DNA cloned into phage orbacterial vectors can be screened by a person skilled in the art. Such alibrary consists e.g. of genomic DNA prepared from human blood cells,fractionized in fragments ranging from 5 kb to 50 kb, cloned into thelambda GEM11 (Promega) phages. Phages hybridizing with the probes can bepurified. From the purified phages DNA can be extracted and sequenced.For example, a human genomic P1 library (Genomic Systems, Inc.) isscreened by a labeled cDNA probe as described in Example 11. Havingisolated the genomic sequences corresponding to the genes encoding theabove-described phosphatonin proteins, it is possible to fuseheterologous DNA sequences to these promoters or their regulatorysequences via transcriptional or translational fusions well known to theperson skilled in the art. In order to identify the regulatory sequencesand specific elements of these phosphatonin genes, 5′-upstream genomicfragments can be cloned in front of marker genes such as luc, gfp or theGUS coding region and the resulting chimeric genes can be transfectedinto cells or animals for transient or stable expression. The expressionpattern observed in the transgenic animals or transfected mammaliancells containing the marker gene under the control of the regulatorysequences of the invention can be compared with that of the phosphatoningene described in Example 10 and reveals the boundaries of the promoterand its regulatory sequences. Usually, said regulatory sequence is partof a recombinant DNA molecule, e.g., a vector see supra. The presentinvention furthermore relates to host cells transformed with aregulatory sequence or a DNA molecule or vector containing theregulatory sequence of the invention. Said host cell may be aprokaryotic or eukaryotic cell; see supra.

[0210] Diagnosing Disorders of Phosphate Metabolism

[0211] Another object of the present invention concerns thepharmacogenomic selection of drugs and prodrugs for patients sufferingfrom disorders in phosphate metabolism (see, e.g., Example 6) and whichare possible candidates to drug therapy. Thus, the findings of thepresent invention provide the options of development of new drugs forthe pharmacological intervention with the aim of restituting thefunction of genetically modified phosphatonin proteins. Also a genetherapeutical approach can be envisaged with the aid of the presentinvention.

[0212] Thus, the invention provides a diagnostic method of a disorder,which involves:

[0213] (a) assaying phosphatonin gene expression level in cells or bodyfluid of an individual; and

[0214] (b) comparing the phosphatonin gene expression level with astandard phosphatonin gene expression level, whereby an increase ordecrease in the assayed phosphatonin gene expression level compared tothe standard expression level is indicative of disorder in phosphatemetabolism, e.g., the kidney or bone system, or other tissues.

[0215] More particularly, the present invention relates to a method ofdiagnosing a pathological condition or a susceptibility to apathological condition in a subject related to a disorder of phosphatemetabolism comprising:

[0216] (a) determining the presence or absence of a mutation in thepolynucleotide encoding phosphatonin; and

[0217] (b) diagnosing a pathological condition or a susceptibility to apathological condition based on the presence or absence of saidmutation.

[0218] In another embodiment, the present invention relates to a methodof diagnosing a pathological condition or a susceptibility to apathological condition in a subject related to a disorder of phosphatemetabolism comprising:

[0219] (a) determining the presence or amount of expression of aphosphatonin polypeptide or a mutant form thereof in a biologicalsample; and

[0220] (b) diagnosing a pathological condition or a susceptibility to apathological condition based on the presence or amount of expression ofthe polypeptide.

[0221] It is evident that the above-described nucleic acid probes andantibodies of the invention are preferably used for the mentionedmethods.

[0222] The above described diagnosis method can also be employed todetermine the status of said disorders. In connection with the presentinvention, the term “pathological condition” include the options thatthe gene, MRNA, protein or a transcription control element, e.g.promoter/enhancer sequence may bear a mutation, deletion or any othermodifications which would affect the overall activity of the gene whencompared to the wild-type normal gene product. Included in this term arepost-translational modifications of the protein.

[0223] In a preferred embodiment of the method of the present inventionsaid status in said subject is indicative of a certain form of thedisorder in phosphate metabolism. Furthermore, it can be advantageousthat in the method of the invention said status in said subject isdetermined in the embryonic status or in the newborn status, for exampleusing aminocentesis.

[0224] The specific analysis of the status of (potential) disorder ofphosphate metabolism at the embryonic, newborn or adult stage willprovide further insights into, e.g., specific disease states associatedwith the respective stages. For example, it is expected that theetiology of, e.g., X-linked Hypophosphatemic Rickets (XHL) or OncogenicHypophosphatemic Osteomalacia (OHO) will be elucidated by applying themethods of the present invention. Upon the basis of this knowledge, newpharmaceutical active drugs will be developed and tested. The method ofthe invention can also be applied to a variety of animals, depending onthe purpose of the investigation. Thus, in a preferred embodiment, theanimal is a mouse. This embodiment is particularly useful for basicresearch to understand more clearly the functional interrelationship ofdifferent proteins which regulate the phosphate metabolism. In a furtherembodiment the animal is a human. In this embodiment, preferablydiagnostic and therapeutic applications are envisaged.

[0225] In a preferred embodiment of the above-described method a furtherstep comprising treating said newborn with a medicament to abolish oralleviate a disorder in phosphate metabolism is performed. Earlydiagnosis of a disorder in phosphate metabolism or susceptibility tothis disorder is particularly advantageous and of considerable medicalimportance. This preferred embodiment can be used to diagnose the statusin, e.g., the coronar villi, i.e. prior to the implantation of theembryo. Furthermore, the status can, with the method of the presentinvention, be diagnosed via amniocentesis. The early diagnosis ofdisorders in the phosphate uptake and/or reabsorption in accordance withall applications of the method of the invention allows treatmentdirectly after birth before the onset of clinical symptoms.

[0226] X-linked rickets patients and tumour osteomalacia patients (priorto tumour resection, or if resection is not possible), are treated withhigh doses of calcitriol or 1,25 dihydroxy vitamin D3 (also knowncommercially as RocaltrolR and is available from Roche; see web site fordetailed information on administrationhttp://www.rochecanada.com/rocaltrol_pml_e.html, and oral phosphatesupplements (dibasic sodium phosphate and/or phosphoric acid). Vitamin Danalogs are also occasionally used (e.g., dihydrotachysterol), andurinary loss of phosphorus and calcium is reported to be further reducedby the additional use of thiazide diuretics such as hydrochlorothiazideand amiloride (Alon, Paediatrics 75 (1985), 754-763). For an extensivereview of current treatments refer to (Carpenter, Pediatric Clinics ofNorth America 44 (1997), 443-466). In children bones need to be reset bybreaking deformed limbs (osteotomy), and the medications described aboveresult in severe vomiting and diarrhea. Growth defects associated withfamilial rickets cannot be satisfactorily addressed using currenttreatments.

[0227] Replacing the above medications with phosphatonin and/orphosphatonin-peptide derivatives would correct the clinical symptoms andnormalize the growth defects without the unpleasant side effects andsurgical osteotomies.

[0228] In another preferred embodiment of the above-described methods,said methods further comprise introducing the functional and expressiblephosphatonin gene into cells of a subject having a disorder orsusceptibility to a disorder in phosphate metabolism. In this contextand as used throughout this specification, “functional” phosphatoningene means a gene wherein the encoded protein having part or all of theprimary structural conformation of the phosphatonin polypeptidepossessing the biological activity described above. The detection of anexpression of a mutant form of phosphatonin would allow the conclusionthat said expression is interrelated to the generation or maintenance ofa disorder in phosphate metabolism. Accordingly, one alternative oradditional step would be applied to reduce the expression level to lowlevels of the mutant phosphatonin or abolish the same. This can be done,for example, by at least partial elimination of the expression of themutant gene by biological means, for example, by the use of ribozymes,antisense nucleic acid molecules or intracellular antibodies against themutant forms of these proteins. Furthermore, pharmaceutical products maybe developed that reduce the expression levels of the correspondingmutant genes.

[0229] Binding Activity

[0230] In a further aspect the present invention relates to a method foridentifying a binding partner to a phosphatonin polypeptide comprising:

[0231] (a) contacting a phosphatonin polypeptide of the invention with acompound to be screened; and

[0232] (b) determining whether the compound effects an activity of thepolypeptide.

[0233] Phosphatonin polypeptides may be used to screen for proteins thatbind to phosphatonin or for proteins to which phosphatonin binds. Thebinding of phosphatonin and the molecule may activate (agonist),increase, inhibit (antagonist), or decrease activity of the phosphatoninor the molecule bound. Examples of such molecules include antibodies,oligonucleotides, proteins (e.g., receptors), or small molecules.

[0234] Preferably, the molecule is closely related to the natural ligandof phosphatonin, e.g., a fragment of the ligand, or a natural substrate,a ligand, a structural or functional mimetic; see, e.g., Coligan,Current Protocols in Immunology 1(2) (1991); Chapter 5. Similarly, themolecule can be closely related to the natural receptor to whichphosphatonin binds, or at least, a fragment of the receptor capable ofbeing bound by phosphatonin (e.g., active site). In either case, themolecule can be rationally designed using known techniques; see alsosupra.

[0235] Preferably, the screening for these molecules involves producingappropriate cells which express phosphatonin, either as a secretedprotein or on the cell membrane. Preferred cells include cells frommammals, yeast, Drosophila, or E. coli. Cells expressing phosphatonin(or cell membrane containing the expressed polypeptide) are thenpreferably contacted with a test compound potentially containing themolecule to observe binding, stimulation, or inhibition of activity ofeither phosphatonin or the molecule.

[0236] The assay may simply test binding of a candidate compound tophosphatonin, wherein binding is detected by a label, or in an assayinvolving competition with a labeled competitor. Further, the assay maytest whether the candidate compound results in a signal generated bybinding to phosphatonin.

[0237] Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining phosphatonin, measuring phosphatonin/molecule activity orbinding, and comparing the phosphatonin/molecule activity or binding toa standard.

[0238] Preferably, an ELISA assay can measure phosphatonin level oractivity in a sample (e.g., biological sample) using a monoclonal orpolyclonal antibody. The antibody can measure phosphatonin level oractivity by either binding, directly or indirectly, to phosphatonin orby competing with phosphatonin for a substrate.

[0239] All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat disease or to bring about a particular result in a patient (e.g.,increase of phosphate level in the blood) by activating or inhibitingthe phosphatonin/molecule. Moreover, the assays can discover agentswhich may inhibit or enhance the production of phosphatonin fromsuitably manipulated cells or tissues.

[0240] Therefore, the invention includes a method of identifyingcompounds which bind to phosphatonin comprising the steps of:

[0241] (a) incubating a candidate binding compound with phosphatonin;and

[0242] (b) determining if binding has occurred.

[0243] Moreover, the invention includes a method of identifyingagonists/antagonists comprising the steps of:

[0244] (a) incubating a candidate compound with phosphatonin;

[0245] (b) assaying a biological activity as described above, and

[0246] (c) determining if a biological activity of phosphatonin has beenaltered.

[0247] As mentioned hereinbefore, the polynucleotides and polypeptidesof the present invention provide a basis for the development of mimeticcompounds that may be inhibitors or activators of phosphatonin or theirencoding genes. It will be appreciated that the present invention alsoprovides cell based screening methods that allow ahigh-throughput-screening (HTS) of compounds that may be candidates forsuch inhibitors and activators.

[0248] In a further embodiment, the present invention relates to amethod of identifying and obtaining a drug candidate for therapy ofdisorders in phosphate metabolism comprising the steps of

[0249] (a) contacting the polypeptide of the present invention or a cellexpressing said polypeptide in the presence of components capable ofproviding a detectable signal in response to phosphate uptake, with saiddrug candidate to be screened under conditions to permit phosphatemetabolism, and

[0250] (b) detecting presence or absence of a signal or increase of thesignal generated from phosphate metabolism, wherein the presence orincrease of the signal is indicative for a putative drug.

[0251] For example, renal cell line CL8, human primary renal cells, orprimary human osteoblast cells can be used to measure radioactiveNa+-dependent phosphate uptake and/or vitamin D metabolism using methodsdescribed by, e.g., Rowe, 1996; supra.

[0252] Furthermore, poly A+ RNA or total RNA extracted from cellsdescribed in (a), and oligonucleotide primers complementary to sequencefor phosphate transporter genes (NPTII etc), renal 24-hydroxylase, a αhydroxylase, PTH, or osteopontin to measure expression of these genesusing, e.g., the polymerase chain reaction can be employed.

[0253] In addition, the measurement of mineralization of human primaryosteoblast cells using von kossa stain is feasible. This methodcomprises, for example,

[0254] growing human primary-osteoblasts (obtainable fromClonetics-Biowhitaker) to confluence using media supplements andconditions recommended by Clonetics;

[0255] for mineralization experiments supplementing the cells withphosphate donor β-glycerphosphate, and for controlshydrocortisone-11-hemisuccinate;

[0256] supplementing experimental cells with β-glycerphosphate and MEPE25 ng/ml;

[0257] After 3 weeks in culture and serial changes of media staining theosteoblasts for bone mineralization using the Von-Kossa stain asdescribed by Clonetics (AgNO₃; silver salt precipitation).

[0258] Furthermore, assays comprising the following measures can beemployed:

[0259] Rat perfusion experiments and measuring effects of phosphatoninon renal phosphate uptake;

[0260] determining the expression of a range of relevant genes inhuman-renal cell line CL8 and the effects of MEPE supplementation, suchas:

[0261] Na+ Phosphate transporters,

[0262] 24 and 1-α hydroxylase,

[0263] Osteopontin and osteocalcin;

[0264] co-transfection system in COS cells with MEPE and PHEX;

[0265] Bio-assay studies using peptide fragments comprising at least oneof the above described motifs. Hence, another detection method comprisesthe measurement of protein kinase C, casein kinase II, tyrosines kinaseor other signal transduction pathways in cells exposed to phosphatoninand derivative peptides using contemporary techniques. Furthermore, themethods as described in the appended examples can be easily adapted tothe above-described screening methods.

[0266] The drug candidate may be a single compound or a plurality ofcompounds. The term “plurality of compounds” in a method of theinvention is to be understood as a plurality of substances which may ormay not be identical.

[0267] Said compound or plurality of compounds may be chemicallysynthesized or microbiologically produced and/or comprised in, forexample, samples, e.g., cell extracts from, e.g., plants, animals ormicroorganisms. Furthermore, said compound(s) may be known in the artbut hitherto not known to be capable of suppressing or activatingphosphatonin polypeptides or other components in the phosphatemetabolism. The reaction mixture may be a cell free extract or maycomprise a cell or tissue culture. Suitable set ups for the method ofthe invention are known to the person skilled in the art and are, forexample, generally described in Alberts et al., Molecular Biology of theCell, third edition (1994) and in the appended examples. The pluralityof compounds may be, e.g., added to the reaction mixture, culturemedium, injected into a cell or otherwise applied to the transgenicanimal. The cell or tissue that may be employed in the method of theinvention preferably is a host cell, mammalian cell or non-humantransgenic animal of the invention described in the embodimentshereinbefore.

[0268] If a sample containing a compound or a plurality of compounds isidentified in the method of the invention, then it is either possible toisolate the compound from the original sample identified as containingthe compound capable of suppressing or activating phosphatonin, or onecan further subdivide the original sample, for example, if it consistsof a plurality of different compounds, so as to reduce the number ofdifferent substances per sample and repeat the method with thesubdivisions of the original sample. Depending on the complexity of thesamples, the steps described above can be performed several times,preferably until the sample identified according to the method of theinvention only comprises a limited number of or only one substance(s).Preferably said sample comprises substances of similar chemical and/orphysical properties, and most preferably said substances are identical.

[0269] The compounds which can be tested and identified according to amethod of the invention may be expression libraries, e.g., cDNAexpression libraries, peptides, proteins, nucleic acids, antibodies,small organic compounds, hormones, peptidomimetics, PNAs or the like(Milner, Nature Medicine 1 (1995), 879-880; Hupp, Cell 83 (1995),237-245; Gibbs, Cell 79 (1994), 193-198 and references cited supra).Furthermore, genes encoding a putative regulator of phosphatonin proteinand/or which exert their effects up- or downstream the phosphatoninprotein of the invention may be identified using, for example, insertionmutagenesis using, for example, gene targeting vectors known in the art(see, e.g., pShooter plasmid series that target expression to thenucleus, mitochondria, or cytoplasm pEF/myc/nuc, pCMV/myc/nuc,pEF/myc/mito, pCMV/myc/mito, pEF/myc/cyto, pCMV/myc/cyto, or pDISPLAYexpression vector that targets recombinant proteins to the surface ofmammalian cells. All the vectors are obtainable from Invitrogen(http://www.invitrogen.com/)).

[0270] Determining whether a compound is capable of suppressing oractivating phosphatonin proteins can be done, for example, by monitoringNa+-dependent phosphate uptake or bone mineralization; see supra. It canfurther be done by monitoring the phenotypic characteristics of the cellof the invention contacted with the compounds and compare it to that ofwild-type cells. In an additional embodiment, said characteristics maybe compared to that of a cell contacted with a compound which is eitherknown to be capable or incapable of suppressing or activatingphosphatonin proteins.

[0271] Once the described compound has been identified and obtained, itis preferably provided in a therapeutically acceptable form. Thus, thepresent invention also relates to a method of producing a therapeuticagent comprising the steps of the methods of the invention describedabove; and

[0272] (i) synthesizing the compound obtained or identified in step (b)of a method of the invention or an analog or derivative thereof in anamount sufficient to provide said agent in a therapeutically effectiveamount to a patient; and/or

[0273] (ii) combining the compound obtained or identified in step (b) ofa method of the invention or an analog or derivative thereof with apharmaceutically acceptable carrier.

[0274] Methods for the preparation of chemical derivatives and analoguesare well known to those skilled in the art and are described in, forexample, Beilstein, Handbook of Organic Chemistry, Springer edition NewYork Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. and OrganicSynthesis, Wiley, New York, USA. Furthermore, said derivatives andanalogues can be synthesized and tested for their effects according tomethods known in the art; see also supra and infra.

[0275] In summary, the present invention provides methods foridentifying compounds which are capable of modulating phosphatemetabolism due to their direct or indirect activation or phosphatonin.Accordingly compounds identified in accordance with the method of thepresent invention to be inhibitors and activators, respectively, ofphosphatonin activity are also within the scope of the presentinvention.

[0276] As is evident from the above, the present invention generallyrelates to compositions comprising at least one of the aforementionedpolynucleotides, nucleic acid molecules, vectors, proteins, regulatorysequences, recombinant DNA molecules, antibodies or compounds.Preferably, said composition comprises ingredients such as buffers,cryoprotectants etc. which are not naturally associated with thementioned components of the invention and render the same suitable for aparticular use.

[0277] Advantageously, said composition is for use as a medicament, adiagnostic means or a kit. Pharmaceutical compositions are described inmore detail in Examples 6 and 7. In particular, bioactive fragments asdescribed above may be useful as a medicament in the treatment of adisorder of phosphate metabolism such as X-linked rickets andosteomalacia as well as other diseases of bone mineral metabolism. Thereis further provided phosphatonin and PHEX metallopeptidase as a combinedpreparation for simultaneous, separate or sequential use as amedicament. In this way, the PHEX metallopeptidase may be used to cleavephosphatonin so as to produce active phosphatonin fragments which may beused for the treatment of disorders of phosphate metabolism as discussedherein. Whilst all of these diseases are particularly important inhumans, other mammals may also be treated in accordance with theinvention.

[0278] The present invention has provided for the first timephosphatonin in a substantially isolated or purified form which issuitably free of contaminants. Native phosphatonin and native fragmentsof phosphatonin, which are free of contaminants such as SDS and/or otherinterfering proteins are capable of regulating phosphate metabolism andof providing active ingredients in pharmaceutical compositions for thetreatment of diseases associated with disorders of phosphate metabolism.

[0279] Hence, the present invention relates to the use of a phosphatoninpolypeptide of the present invention or a DNA encoding and capableexpressing said polypeptide, the antibody, the activator/agonist,inhibitor/antagonist or binding partner of the present invention, forthe preparation of a medicament for treatment of a disorder of phosphatemetabolism.

[0280] In particular, the present invention relates to the use of aphosphatonin polypeptide having phosphatonin activity or a DNA encodingand capable expressing said polypeptide, the antibody, theactivator/agonist or binding partner of the invention whose presence inthe cell leads to phosphatonin activity, for the preparation of amedicament for the treatment of hyperphosphatemia, preferably for thetreatment of renal osteodystrophy, hyperphosphatemia in renaldialysis/pre-dialysis, secondary hyperparathyrodism or osteitis fibrosacystica.

[0281] In another embodiment, the present invention relates to the useof a phosphatonin polypeptide having anti-phosphatonin activity or a DNAencoding and capable expressing said polypeptide, the antibody of theinvention, the nucleic acid molecule or the inhibitor/antagonist of thepresent invention, for the preparation of a medicament for the treatmentof hypophosphatemia, preferably for the preparation of a medicament forthe treatment of X-linked hypophosphatemic rickets, hereditaryhypophosphatemic rickets with hypercalcuria (HHRH), hypomineralized bonelesions, stunted growth in juveniles, oncogenic hypophosphatemicosteomalacia, renal phosphate leakage, renal osteodystrophy,osteoporosis, vitamin D resistant rickets, end organ resistance, renalFanconi syndrome, autosomal rickets, Paget's disease, kidney failure,renal tubular acidosis, cystic fibrosis or sprue.

[0282] In a preferred embodiment of the present invention, thephosphatonin polypeptide having anti-phosphatonin activity or a DNAencoding and capable expressing said polypeptide, the antibody of theinvention, the nucleic acid molecule of the invention or theinhibitor/antagonist of the invention are used for the manufacture of amedicament for the treatment of a bone mineral loss disorder.

[0283] In another preferred embodiment, the present invention relates tothe use of a phosphatoninpolypeptide and PHEX metallopeptidase for themanufacture of a combined preparation for simultaneous, separate orsequential use for the treatment of a disorder of phosphate metabolism.

[0284] The above-mentioned uses and methods are described in more detailin Example 6.

[0285] In another embodiment, the present invention relates to the useof a transformed osteoblast or bone cell line capable of phosphatoninoverexpression for the production and isolation of phosphatonin.

[0286] The following examples are put forth so as to provide thoseskilled in the art with a complete disclosure and description of how tocarry out various aspects of the invention and are not intended to limitthe scope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. Efforts have been made to ensureaccuracy with respect to numbers used (e.g., amounts, temperatures,etc.) but some experimental error and deviation should be accounted for.Unless indicated otherwise parts are parts by weight, molecular weightis weight average molecular weight and temperature is in degreescentigrade.

EXAMPLE 1 Purification of Phosphatonin from Tumor

[0287] A mesenchymal tumor with phosphaturic expression was removed froma patient and the following samples taken:

[0288] A: Sample of pure tumor tissue, size of two large peas, wasplaced into a 2 ml vial containing DMEMEagles/10%/oFCS/glutamine/antibiotic antimycotic Gibco-BRL.

[0289] B: Sample of sub-dura tumor approximately the same size possiblysmaller. Placed in same media as A.

[0290] C: Sample of abnormal dura: tough white material: Placed in samemedia as A.

[0291] D: Sample of tumor fluid.

[0292] Processing of Samples:

[0293] Day 1:

[0294] The samples were each cut into small 0.5 cm cubes using a sterilescalpel. Half of each sample was placed into a cryotube and frozen downin N2(1) immediately. The fluid surrounding the tissue (DMEM/10% FCSetc.), was also collected and frozen down. The other half of each samplewas added to DMEM Eagles/10% FCS/glutamine/antimycotic antibioticsupplemented with collagenase Al 0.2 mg/ml (˜15 ml), and left at 37° C.overnight.

[0295] Day 2:

[0296] 1. After overnight incubation in serum supplemented DMEM, thecells appeared to be predominantly RBC's and very few adherent cellswere observed. The cells were spun down at room temp and thesupernatants collected and immediately frozen down (˜15 ml).

[0297] 2. The pellets were then resuspended in 10 ml of DMEM Eaglessupplemented with antibiotic/antimycotic (medium flasks), and thenincubated for a further 8 h 10 min.

[0298] 3. The serum-free supernatants were collected as described in 1(˜10 ml), and the cells were resuspended in DMEM EAGs with 10% FCS etc.,(˜15 ml), and incubation continued. The supernatants were stored at −80°C.

[0299] Day 6:

[0300] 1. After incubation from Day 2, cells were spun down as describedfor I of Day 2. 10% FCS samples were collected and frozen.

[0301] 2. Pellets were resuspended in serum free DMEM (10 ml), as forDay 2 and this time left for four hours.

[0302] 3. Same as for 3 of Day 2.

[0303] Day7:

[0304] 1. The subdura and tumor culture in particular, had developedinnumerable foci containing clumps of cells which appeared attached tothe plastic of the tissue culture plates. Underneath these polyp likeprotuberances was a monolayer of fibroblast like cells which spread outradially from underneath the tumor like structures. This layer of cellsappeared to act as a matrix to anchor the polyp like tumors. None ofthis was seen in the dura sample, which appeared to lack cells at thisstage, and contained fibrous like matted structures.

[0305] 2. Cultures were spun down, and the supernatants collected (10%FCS). The pellets were then placed to one side.

[0306] 3. The plates were then incubated with 10 ml of trypsin EDTA solnGibco/BRL 1/10 dilution in PBS for −15 min. Plates were then tappedvigorously and 5 ml of FCS added.

[0307] 4. The resuspended cells were then added to the pellets obtainedin 2, resuspended and spun down. The supernatant was discarded.

[0308] 5. Cells were then plated out in 18 ml of 10 % FCS DMEM Eaglesmedium with glutamine and antibiotic antimycotic supplements (largeflakes were used.)

[0309] 6. Finally cells were incubated at 37° C. in CO₂ atmosphere.

[0310] Day 9:

[0311] 1. Tumor cells and to some extent the subdura cells appeared asinnumerable clumps of cells, and appeared to have the same morphology asthe cells prior to trypsin treatment. Some of the clumps were quitelarge, and visible to the naked eye.

[0312] 2. The serum supplemented media was collected and stored down.Large flasks were used and 18 ml of media per flask added (DMEM 10% FCSantimycotic/antibiotic/glutamine).

[0313] Day 13:

[0314]1. Cells were frozen down (−15 ml), and stored in falcons as 10%FCS DMEM conditioned media.

[0315] 2. Cells resuspended in serum free DMEM Eags (−11 ml) 11.10 am,and left for 6 h at 37° C. (CO₂ incubator).

[0316] 3. Cells were then spun down and the supernatants collected(serum free control media). 10% FCS DMEM EAG was then added to theremaining cells.

[0317] Day 16:

[0318] The above process was repeated and Tumor Conditioned Medium (TCM)collected over several weeks. Alternatively, TCM may be collected fromSaos-2 cells (ECACC 89050205) or U-2 OS cells (ATCC HTB-96).

[0319] Purification of Phosphatonin:

[0320] Concanavalin A Sepharose Affinity Chromatography:

[0321] 1. 3 ml of TCM was adjusted with IM sodium phosphate pH 7.2 andSM NaCl to give a final concentration of 0.06M Sodium phosphate pH 7.2and 0.5M NaCl plus 0.01% sodium azide.

[0322] 2. Con A Sepharose (Pharmacia Code No: 17-0440-01), arrived in20% Ethanol, and this was first washed with several column volumes ofwater, and then equilibrated in the running buffer. A small C10/10column (Pharmacia code No: C10/10 id 10 mm), was packed with Con A to aheight of 5.5 cm (approx. volume 4.3 to 5.0 ml). Equilibration wascarried out at max flow rate of 0.5 ml/min.

[0323] 3. The sample (adjusted to pH 7.2 sodium phosphate/0.5MNaCl/0.01% sodium azide), was then added to the column by gravity feed,and reloaded three times. The color of the sample enabled visualizationof the passage through the column. Unbound material was then collectedand stored for future reference.

[0324] 4. Waters LC system was then connected and the sample was washedwith several column volumes of loading buffer.

[0325] 5. After loading and washing, elution was carried out usingsodium phosphate buffer 60 mM pH 7.2/0.5M Nacl/0.5Mα-methyl-D-glucopyranoside/0.01% azide buffer. See FIG. 1a. A singlepeak was detected and this was collected.

[0326] 6. The column was then run to base line approximately 40 ml max,and then left overnight.

[0327] 7. After overnight incubation in methyl glycoside buffer, asecond peak was eluted (see FIG. 1b), which peaked at −5 ml.

[0328] 8. The second peak was collected and dialyzed against 0.05Macetic acid, and then lyophilized. Both Concanavalin peaks Al (lowaffinity), and concanavalin A2 (high affinity), are potent at inhibitingNa+ dependent phosphate co-transport and vitamin D metabolism in a humanrenal cell line (CL8). The high affinity fraction, the human renal cellline (CL8), and the conditions used for assay are described in Rowe etal 1996. A further suitable known renal cell line for this assay is theOK cell line deposited as ECACC 91021202.

[0329] Cation Exchange Chromatography using HiTrap SP Cation Exchange 1ml Column (Code No 17-1151-01; Pharmacia):

[0330] 1. The lyophilized protein was then re-dissolved in 0.05Mammonium acetate pH 5 and the applied to an equilibrated 1 ml HiTrap SPsepharose cation exchange column.

[0331] 2. The column was equilibrated prior to sample addition bywashing with water, and then 5 volumes of start buffer (0.02 M ammoniumacetate pH 5).

[0332] 3. Sample was eluted using the following protocol; Time Flow rate% NH₄ acetate % NH₄ acetate/ Num Min ml/min pH 5 0.5 M NaCl pH 5 1 0.5100  0 2 15 0.5  25  75 3 20 0.5  0 100 4 25 0.5  0 100 5 35 0.5 100  06 50 0.5 100  0

[0333] A Single sharp peak was obtained, and the sample was thendialyzed against 0.05M acetic acid and lyophilized; see FIG. 2.

[0334] After resuspending in 10 mM phosphate buffer pH 7.2 20 μl,aliquots were resuspended in SDS-PAGE sample buffer (to a finalconcentration=125 mM TRIS-HCL pH6; 2.5% glycerol; 0.5% w/v SDS: 5%β-mercaptoethanol; 0.01% bromophenol blue), boiled (5 mins), cooled andthen run on an SDS PAGE gel 12.5% (see chromatogram), and a double bandof 55 kD was resolved (see Rowe et al 1996). Both the Concanavalin A andcation bands also have an aggregated form. All fractions including thetumor conditioned media were potent at inhibiting Na+ dependentphosphate co-transport in a human renal cell line (1/1000 diln), andalso altered vitamin D metabolism. For a full description of the methodsused to measure phosphate transport and vitamin D metabolism see Rowe etal 1996. All purification modalities were carried out on a watersHPLC/FPLC system programmed by computer-millennium software. The mostactive fraction was the concanavalin Al fraction from OHO tumor. Antipre-operation antisera was used to screen the immobilized purifiedfraction. The fraction is also potent at inhibiting NaPi, and affectsvitamin D metabolism in a human renal cell line (CL8).

EXAMPLE 2 Screening of Tumor Conditioned-medium (TCM), and PurifiedFractions with Pre/Post- Operation Antisera; Plus Glycoprotein Screen

[0335] Pre-operation and post-operation antisera from a patient has beendescribed previously in Rowe et al 1996. Only pre-operation antiseradetected the purified fractions and hormone in TCM in which Western andglycoprotein detection of TCM and purified fractions was achieved usingenhanced chemiluminescence. Protein markers were biotinylated, andtagged with streptavidin peroxidase conjugate. The arrows show theaggregate and active glycoprotein. Post-operation antisera and rabbitpre-immune sera did not detect any of the fractions. Also, only thosetumors secreting phosphaturic factor were positive. Media and skincontrols were negative. A distinct feature of the Con A1, Con A2 and CA1samples was their potent ability to inhibit NaPi, and alter vitamin Dmetabolism in a human renal cell line (CL8). All the purified fractionshave a tendency to aggregate into a lower mobility form on SDS-PAGE.Also, the purified fractions and TCM active fractions are heavilyglycosylated. The extent of glycosylation was confirmed by periodateoxidation of immobilized proteins on PVDF membranes followed bybiotinylation of carbohydrate moieties. These were then screened withstreptavidin conjugated to horse radish peroxidase and enhancedchemiluminescence. The active form (inhibits NaPi etc.), is associatedwith the 58 to 60 kDa fraction. An additional and powerful way ofpurifying the protein to homogeneity is the use of a neutral pH 7SDS-PAGE system using a 4-12% Bis-Tris Gel with MOPS running buffer.Pre-caste gels can be purchased from Novex.

EXAMPLE 3 SDS-PAGE at Neutral pH using 4-12% Polyacrylamide Gradient andBis-Tris Gel with MOPS Running Buffer (Nu-PAGE System from NOVEX):Reduced Mobility of Hormone

[0336] On this system a fraction of the glycosylated hormone has areduced mobility, and runs at ˜200 kDa. The lower molecular weight formis also visible at 58/60 kDa. Appearance of the ˜200 kDa protein may bedue to the isoelectric point of the protein (different charge at neutralpH), and the interaction of carbohydrate moiety with the gel matrix.Also, increased efficiency of electro-blotting of high molecular weightcomponents occurs due to the low % acrylamide (4-12% gradient), at thetop of the gradient gel. Running fractions through this system increasesthe purity and homogeneity of the molecule. A Western blot using thissystem and including the following samples (pre-operation antiserum wasused to screen the blots using enhanced chemiluminescence detection): 1.protein markers; 2. intracranial tumor cell line OHO; 3. cells fromsub-dura adjacent to tumor; 4. cells from dura adjacent to sub-dura; 5.HTB6 cell line; 6. Saos-2 cell line; 7. defined medium control; 8. Skinfibroblast control; 9. Linear sebaceous naevus polyp tumor demonstratedthat Naevus polyp tumor showed a specific phosphaturic band at ˜200kDaon SDS-PAGE Neutral gels.

EXAMPLE 4 Cloning and Sequencing of Phosphatonin

[0337] 1. Library Construction:

[0338] A tumor derived from a patient described in an earlierpublication (BD, Rowe et al., 1996), was sectioned and mRNA extractedusing standard techniques. The mRNA was copied using reversetranscriptase to generate a cDNA population that was then subsequentlysubcloned into a bacteriophage vector λ-ZAP II uni (vector purchasedfrom Stratagene Ltd., Unit 140, Cambridge Science Park, Milton Road,Cambridge, CB4 4GF United Kingdom). The cloning was uni-directional andthe 5′ end of the gene was adjacent to the T3 promoter and abutted anEcoRI site. The 3′ end of the cDNA's abutted an Xho-1 site upstream of abacterial T7 promoter. Briefly, resected tumour from patient BD was cutinto 1 mm blocks and poly A+ RNA extracted directly usingStreptavidin-Magnesphere paramagnetic particle technology (PolyATract®system Promega). The purified mRNA was then used to generate a cDNAtemplate using the cDNA synthesis kit from Stratagene. Linker primerswere added to the CDNA to generate a 5′ EcoRI compatible CDNA end, andan XhoI compatible 3′ cDNA end, to facilitate forced orientation cloninginto AZAP II uni bacteriophage vector. Recombinant bacteriophages wereplated out and mplified on E. coli XLl-Blue mrf′. Total primary clonesnumbered 800000 with 6% wild type representation.

[0339] 2 . Screening with Pre-operative Antisera:

[0340] The cDNA bacteriophage library was plated out of NZY agar platesand the β-galactosidase operon induced using IPTG. Expressed fusionproteins were then transferred to hybond-C membranes (Amersham) and themembranes were then screened with pre-operation antisera from thepatient. The antisera used has been described (Rowe et al., 1996). Priorto use the antisera was extensively pre-absorbed with E. coli lysate,and whole blood to reduce signal to noise. Rabbit antisera raisedagainst patient BD pre-operation serum (Rowe, Bone 18 (1996), 159-169),was extensively pre-absorbed with normal human serum and E. coli lysatein order to remove E. coli antibodies and background human-serum derivedantibodies. Briefly, five 80 mm diameter nitrocellulose filters wereadded to whole E. coli lysate (Stratagene), and a second set of fivefilters were soaked with normal human serum (10 ml). The impregnatedfilters were each incubated for 10 min at room temperature in sequencewith 250 ml of 1:1000 diluted anti rabbit pre-operation antisera in 1%BSA; 20 mM Tris-HCl (pH7.5), 150 mM NaCl (TBS); 0.02% NaN₃. Thepreabsorbed pre-operation antisera (pre-Aanti-op) was then used toscreen the cDNA library. Bacteriophage λZAP II uni OHO cDNA-clones wereplated out on E. coli XL1-Blue mrf′ and incubated for 3 hours at 37° C.Hybond N⁺ filters preincubated with 10 mM IPTG were then placed on topof the developing plaques and incubated a further 3 h at 42° C. Filterswere then removed and washed with TBS supplemented with Tween 20 (TBST),and then blocked with 1% BSA in TBS with 0.02% NaN3 overnight at 4° C.Pre-Aanti-op was then added to the blocked filters and left for 1 h atroom temperature. Subsequent washes of the filters and incubation withgoat-anti-rabbit alkaline phosphatase conjugate, followed byvisualization using 5-bromo-4-chloro-3-indolyl phosphate/nitrobluetetrazolium was as described by Stratagenes picoblue™ immunoscreeningkit. After screening ˜600,000 clones, nine positives were selected andpurified by secondary and tertiary screening. The bacteriophage cloneswere rescued as phagemids using ExAssist helper phage and cloned into E.coli SOLR cells. ExAssist helper phage and SOLR cells were purchasedfrom Stratagene Ltd., Suite 140, Cambridge Science Park, Milton Road,Cambridge, CB4 4GF, United Kingdom.

[0341] 3. Sequencing Clone:

[0342] Phagemids were prepared and the DNA sequenced. All nine cloneswere sequenced. Positive bacteriophage-plaques were removed from agaroseplates after tertiary screening with a sterile hollow quill. The agaroseplugs containing the lytic plaques was then added to 0.5 ml of SM buffersupplemented with 0.02% chloroform, and left at 4° C. overnight. Rescueand transformation of bacteriophage clones to BSCPT SKII- phagemids wascarried out using ExAssist phage as described by Stratagene. The hostcells for the purified phagemid were E. coli SOLR cells. Plasmid DNA wasthen prepared using standard techniques (Rowe, Nucleic Acids Res. 22(1994), 5134-5136), and sequenced using ABI fluorescent automatedsequencing and standard vector specific primers. Six of the clones wereoverlapping and in fiame with the bacterial β-galactosidase promoter togive contiguous/overlapping epitopes and expressed proteins withidentical overlapping DNA sequences. The longest sequenced cloneencompassed the cDNA sequences of the five others and is shown in FIG.8. This sequence (amino acid/cDNA) is a complete sequence forphosphatonin. There are 430 amino acid residues cloned (SEQ ID NO: 2)and 1655 bp of DNA sequence (SEQ ID NO: 1). Secondary structureprediction indicates a highly hydrophilic protein with glycosylation atthe COOH end, and the presence of a cell attachment tripeptide at theamino end (RGD), see FIG. 8. The protein is also highly antigenic with anumber of major helical domains (FIG. 10). Extensive screening of allavailable databases using BLAST has not-revealed any statisticallyrelevant homology to known genes or protein sequences.

[0343] 4. Purification of Recombinant Human Phosphatonin:

[0344] The isolated cDNA clone is represented as rescued phagemids inBscpt SKII—vector (Stratagene vector), and contained within SOLR E. colihost cells. Low level fusion protein expression via induction of theβ-galactosidase promoter by IPTG has been achieved. The phosphatoninclone fusion-product reacts with pre-operation antisera on westernblots. Increased expression and bioactivity of the fusion proteins canbe achieved by sub-cloning into the pCAL-n-EK vector (Stratagene vector)(see below). The construct containing human phosphatonin is contained inE. coli (BL21 (DE3) pLysS) cells (purchased from Stratagene). IPTGinduction of fusion protein is much higher, and essentially pure proteincan be obtained by calmodulin affinity-chromatography of cell lysates.Recombinant phosphatonin with filsion-tag binds to the calmodulin resinin the presence of Ca²⁺. Phosphatonin fusion protein is then releasedafter washing with EGTA. The small microbial fusion-tag is removed bytreatment with enterokinase, leaving pure human phosphatonin.

[0345] 4a. Subcloning Phosphatonin into pCAL-n-EK Vector

[0346] The entire deduced cDNA coding sequence (deduced from the largestcDNA clone pOHO11.1), of phosphatonin (MEPE) was subcloned into theprokaryote expression vector plasmid pCAL-n-EK (Stratagene vector), andthe construct transformed into E. coli BL21 (DE3) pLysS and E. coliXL1-Blue mrf′ respectively (strains obtained from Stratagene). Themethod of ligation independent cloning (LIC) was used as described byStratagene Affinity™ cloning and protein purification kit (cat No:#214405 and #214407). Two primers were designed from the phosphatoninsequence 5′ and 3′ end respectively with additional overhang linkersequence as follows (bold sequence represents linker): Forward (SEQ IDNO:8) 5′ GACGACGACAAG.GTGAATAAAGAATATAGTATCAGTAA 3′      Linker Reverse(SEQ ID NO:9) 5′ GGAACAAGACCCGT.CTAGTCACCATCGCTCTCACT 3′      Linker

[0347] PCR amplification of phosphatonin includes DNA sequence codingfor the first valine residue to the stop codon of phosphatonin (see FIG.8), plus linker sequence. A 5′ overhang of the linker sequence is thengenerated by treating the PCR fragment with Pfu polymerase and dATP.Induction of fusion protein is carried by growing the cells and addingIPTG. The PCR conditions were as follows. Predenaturation; 95° C. 3min,followed by 20 cycles of Denaturation; 95° C. 45 sec, Annealing 59° C.60 sec, 72° C. 2 min, and then 72° C. 7 min final extension; followed bycooling 4° C. A Perkin Elmer 9600 thernocycler was programmed to carryout the PCR, and the following PCR buffer (PB), was used: 10 mM Tris-HClpH 8, 50 mM KCl, 1 μM primers, 200 μM dNTP's. PB buffer was supplementedwith 2 mM MgCl₂. For ligation independent cloning (LIC), the amplifiedproduct was then treated with pfu polymerase and dATP as described byStratagene, and then directly annealed to linearized pCAL-n-EK plasmidvector with complementary linker overhangs. The construct was thentransformed into competent E. coli XL1-blue mrf′ cells, and competent E.coli BL21 (DE3) Clones were then selected on ampicillin plates, andplasmids prepared and sequenced. A summary of the vector and fusionconstruct is shown in FIG. 14. High copy number plasmid is achieved withE. coli XL1-blue mrf′ host, and high recombinant protein expression isobtained with E. coli BL21 (DE3).

[0348] 4b. Purifying Phosphatonin by Calmodulin Affinity Resin

[0349] The method as described by Stratagene (cat˜214405), can be used.Sequence upstream from the phosphatonin specific residues will contain acalmodulin binding sequence. Calmodulin resin is added to the crude celllysate in the presence of calcium, and the protein allowed to bind. Theslurry is then washed with calcium containing buffer, and thephosphatonin fusion protein eluted by addition of EGTA 2 mM in a Trisbuffer (50 mM Tris-HCI pH 8). Removal of the calmodulin binding proteintag is then accomplished by digestion with site-specific protease EK,leaving pure recombinant human phosphatonin. Preferably, the method maybe performed as follows (See table below for buffer compositions):

[0350] 1. Cells are cultured and induced as described by the Stratageneprotocol for pCAL-n-EK vectors (Cat No: #214405), using BL23 (DE3) E.coli host cells comprising plasmid p1BL21; see FIG. 14.

[0351] 2. Protein lysate is also prepared as described by the Stratageneprotocol but using CCBB-II as resuspension buffer (resuspend cell pelletfrom 500 ml in 10 ml of CCBB-II). It is essential to sonicate in 30 secpulses followed by 4 min cooling with ice. Tubes containing cells arekept on ice during sonication.

[0352] 3. After sonication cells are spun at 10000 g and the supernatantdecanted. Most of the recombinant MEPE remains in the supematant(protein-lysate).

[0353] 4. The protein-lysate is then concentrated by using a VIVASCIENCEVIVASPIN (Cat No: VS1521 called 30,000 MWCO PES) concentrator with a30000 molecular weight cut off. Approximately 8 ml of supernatant from500 ml of cells concentrates down to 3.2 ml (X2.5 conc.). Furtherconcentration is not advisable.

[0354] 5. For protein-lysate prepared from 190-200 ml of cells (˜1.3 mlof equivalent protein-lysate), 1 ml of equilibrated calmodulin resin isthen added (equilibrate resin as described by Stratagene using CCBB-IIbuffer).

[0355] 6. The suspension is rotated overnight at 4° C.

[0356] 7. The suspension is spun down (˜3000 rpm on eppendorf centrifugefor 2 min), the supernatant removed and the resin resuspended in 1 ml ofCCBB-II buffer.

[0357] 8. The resin is spun down again and the first wash removed. Thisis repeated twice more (total of three washes in CCBB-II).

[0358] 9. It is then washed once with WB-III; note non of the buffersincluding the final wash buffer contain detergents. The cells used forbio-assay are extremely sensitive to detergents even in trace amounts.WB-III is the same as CCBB-II but without protease inhibitors.

[0359] 10. Non-specific proteins are eluted by washing with buffer EB-Itwice (1 ml).

[0360] 11. MEPE is eluted with EB-II 2-3 times (1 ml).

[0361] 12. Protein is concentrated using a flowgen 10K microsepconcentrator at 4° C. Generally 3 ml of MEPE eluate can be concentrateddown to ˜170 μl in 2 hr.

[0362] 13. After running samples on an SDS-PAGE gel to assess purity andquantity multiple aliquots are made and frozen at −80° C. Repeatedfreeze thaw is avoided.

[0363] Buffers: Component CCBB-II WBIII EBI EBII Tris0Buffer pH 8 50 mM50 mM 50 mM 50 mM NaCl 300 mM 300 mM 150 mM 1 M MgAcetate 1 mM 0 0 0Imidazole 1 mM 0 0 0 CaCl2 2 mM 2 mM 0 0 Protease YES No No NoInhibitors w/o EDTA EGTA 0 0 4 mM 4 mM

[0364] Protease inhibitor tablets were added 1 per 10 ml when used(Boehringer Mannheim), protease inhibitor w/o EDTA (Cat No: 1836 170). Afinal elution with 1M NaCl, EGTA (4 MM) buffer results in >95% purity ofphosphatonin.

EXAMPLE 5 Structure of Phosphatonin

[0365] 1. Primary Structure and Motifs:

[0366] The primary structure of the protein and the nucleic acidsequence are shown in FIG. 8. The largest cDNA clone isolated for MEPEwas 1655 bp and contained the entire 3′ end of the gene with poly A⁺tail and a single polyadenylation sequence (AA[T/U]AAA) (FIG. 8). Anopen reading frame of 430 residues was found that overlapped andextended the other smaller MEPE cDNA clones isolated, with a predictedM??47.3 kDa and a pI of 7.4. The best fit consensus start codon Kozak,Nucleic Acids. Res. 15 (1987), 8125-8148), occurs at 255 bp, althoughtwo other methionines preceded this. It is possible that additional 5′sequence is missing, and an earlier start codon and or extended 5′untranslated sequence needs to be characterized. GCG- secondarystructure prediction indicates that the protein is very hydrophilic withthree localized areas of low hydrophobicity (FIG. 9). The protein hasglycosylation motifs at residues 382 and 385 (NNST), and residues383-386 (NSTR). There is also a glycosaminoglycan attachment site atresidues 161-164 (SGDG). The approximate molecular weight withoutglycosylation is 54 kDa, and is in close agreement with the purifiedglycosylated form of (58-60 kDa). There are a number of phosphorylationsite motifs (see Table 1), and these are predicted to play a role in thebiological activity of the hormone or fragments thereof. TABLE 1 Site(on FIG. 8) Motif Protein Kinase C phosphorylation  8-10 SNK 77-79 TPR118-120 THR 203-205 TKK 228-230 TAK 311-313 STR 312-314 TRK 319-321 SNR384-386 STR 403-405 SNR 408-410 SSR 409-411 SRR Casein Kinase IIphosphorylation  8-11 SNKE 139-142 SDFE 177-180 TGPD 194-197 SEAE199-202 THLD 224-227 TRDE 228-231 TAKE 238-241 SLVE 325-328 TLNE 423-426SSSE 425-428 SESD 427-430 SDGD Camp- & cGMP-dependent protein 405-408RRFS kinase phosphorylation Tyrosine Kinase Phosphorylation 40-47KLHDQEEY Myristoylation 16-21 GLRMSI 143-148 GSGYTD 119-224 GNTIGT266-271 GSQNAH 291-296 GSSDAA 315-320 GVDHSN 389-394 GMPQGK Amidation370-373 HGRK RGD 152-154 RGD Gycosaminoglycan Attach. Site 161-165 SGDGAsu-Glycosylation 382-386 NNST 383-387 NSTR

[0367] A key feature of the protein is a cell attachment sequence atresidues 152-154 (RGD). The Arg-Gly-Asp sequence plays a role inreceptor interactions in general, and in fibronectin is essential forcell surface receptor binding to a specific integrin. More notable isthe presence of this motif in some forms of collagens (bone matrixprotein), fibrinogen, vitronectin, von Willebrand factor (VWF), snakedisintegrins, and slime mould discoidins. It is highly probable thatthis part of the phosphatonin is involved in receptor and/or bonemineral matrix interactions.

[0368] Also these interactions mediate the following:

[0369] 1. osteoid mineralization (osteoblasts).

[0370] 2. Na-dependent phosphate co-transporter gene expressionregulation.

[0371] 3. 24 hydroxylase and/or 1 alpha hydroxylase gene expressionregulation (kidney).

[0372] 4. bone and dental mineral matrix interactions and regulation ofmineral deposition via nucleation.

[0373] The presence of a glycosaminoglycan attachment sequence atresidues 161-164 (SGDG), has important implications concerning bonemineral attachment and interactions. The role of proteoglycans in boneis well documented particularly in cell signaling. It is highly probablethat this part of the molecule is also essential for the abovebioactivities (point 1 to 4), and in particular osteoblast mediatedmineralization of osteoid.

[0374] The RGD motif is in a region of predicted turn (Gamier predictionAntheprot), and is flanked by two regions of ?-sheet (residues 134 to141 and 172 to 178). The predicted sheet structure is in turn flanked bytwo regions of extended ?-helix (121 to 132 and 196 to 201). The generalstructural context, predicted turn and presence of the RGD cellattachment sequence is similar to that found in osteopontin. The proteinalso has a number of predicted phosphorylation motifs for protein kinaseC, casein kinase II, tyrosine kinase, and cAMP cGMP-dependent proteinkinase. MEPE was also found to have a large number of N-myristoylationsites, and these sites appear to be a feature of RGD containing phosphoglycoproteins (osteopontin, vitronectin, collagen, h-integrin bindingprotein, dentin-sialophosphoprotein, dentin-matrix-protein-1,bone-sialoprotein-II and fibronectin). There is an unusually highcontent of aspartate, serine and glutamate residues (26%), as inosteopontin (37%). Of particular interest is the complete absence ofcysteine residues in MEPE sequence, indicating that cysteine-cysteinedisulphide bridges do not play a role in the secondary structure of thismolecule. Sequence homology to dentin phosphoryn (DPP) was found afterscreening the trembl database with MEPE. A region at the C-terminus ofMEPE has a sequence of aspartate and serine residues (residues 414-427)that are almost identical (80% homology), to a recurring motif found inDPP) (FIG. 26A and 26B). Physicohemical comparison of the MEPE motif(DDSSESSDSGSSSESD) with the DSP motif (SDSSDSSDSSSSSDSS), increases thehomology to 93%. The MEPE-motif occurs once at the C-terminus in MEPE(residues 414 to 427), whereas the DSP homologue is repeated at DSPresidue positions 686 to 699, 636 to 646, and 663 to 677. Moreover, tworelated sequences DSSDSSDSNSSSDS and DSSDSSDSSNSSDS, also with 80%homology to the MEPE-motif are found in DSP at positions 576 to 589 and800 to 813 respectively. A similar motif with 60% homology(DDSHQSDESHHSDESD), is also found in osteopontin (residues 101 to 116),and a casein kinase II phosphorylation site is contained within theregion of homology (FIG. 12). Skeletal casein kinase II activity isdefective in X-linked rickets (Rifas, loc. cit.). Although theosteopontin MEPE-motif is central and not C-terminal, cleavage ofosteopontin in vivo has been reported and this would generate a peptidewith the MEPE motif placed C-terminal (Smith, J. Biol. Chem. 271 (1996),28485-28491). Additional sequence homology to the C-terminal MEPE-motifis also found in DMA-1 at residues 408 to 429 (SSRRRDDSSESSDSGSSSESDG).A graphical presentation of the regional sequence homology of theMEPE-motif in DSSP, DMA-1 and OPN is presented in FIG. 12 as a‘llanview’ statistical plot, and Table. 2 presents the sequencesimilarities in alignment. TABLE 2 MEPE versus DSSP Upper sequence MEPE:414 DSSESSDSGSSSES 427 (SEQ ID NO:7) 686 DSSDSSDSSSSSDS 699 (SEQ IDNO:13) 414 DSSESSDSGSSSES 427 (SEQ ID NO:7) 633 DSSDSSDSSSSSDS 646 (SEQID NO:13) 413 DDSSESSDSGSSSES 427 (SEQ ID NO:10) 551 DDSSDSSDSSDSSDS 565(SEQ ID NO:14) 414 DSSESSDSGSSSES 427 (SEQ ID NO:7) 576 DSSDSSDSNSSSDS589 (SEQ ID NO:15) 414 DSSESSDSGSSSES 427 (SEQ ID NO:7) 663DSSDSSDSSSSSDS 677 (SEQ ID NO:13) 414 DSSESSDSGSSSES 427 (SEQ ID NO:7)752 DSSESSDSSNSSDS 765 (SEQ ID NO:16) 414 DSSESSDSGSSSES 427 (SEQ IDNO:7) 800 DSSDSSDSSNSSDS 813 (SEQ ID NO:17) MEPE versus Osteopontin:Upper sequence MEPE 413 DDSSESSDSGSSSESD 428 (SEQ ID NO:11) 101DDSHQSDESHHSDESD 116 (SEQ ID NO:18) Osteopontin versus DSSP: Uppersequence osteopontin 106 SDESHHSDESD 116 (SEQ ID NO:19) 638 SDSSSSSDSSD648 (SEQ ID NO:20) 106 SDESHHSDESD 116 (SEQIDNO:19) 846 SDSSDSSDSSD 857(SEQ ID NO:21) 106 SDESHHSDESD 116 (SEQ ID NO:19) 857 SDSSDSSDSSN 878(SEQ ID NO:22) MEPE versus DMA-1 MEPE top sequence 408SSRRRDDSSESSDSGSSSESDG 429 (SEQ ID NO:12) 443 SSRSKEDSN-STESKSSSEEDG 463(SEQ ID NO:23)

[0375] Of interest is the repetitive occurrence of the motif at theC-terminal region of DSSP or the dentin-phosphoryn portion. A dot-matrixsequence-comparison of MEPE against DSSP at high and low stringency isshown in FIG. 13, and this illustrates the repetitive occurrence of theaspartate-serine rich MEPE motif in DSSP.

[0376] DPP is formed by post-translational cleavage of a much largerprotein, dentin sialo-phosphoprotein (DSSP), into two distinct proteinsDPP and dentin sialoprotein (DSP). There is considerable sequencehomology of MEPE and osteopontin to the dentin phosphoryn (DPP), part ofdentin siaolo-phosphoprotein (DSSP), with no homology to the dentinsiaolprotein portion of the molecule (DSP) (FIG. 13). Of note is theclose alignment of the RGD motif, casein kinase II phosphorylationmotifs and N-glycosylation sites in both DPP and MEPE (FIG. 13). Also,all the protein kinase C sites associated with DSSP are clustered in theregion of overlap with MEPE (dentin phosphoryn portion), with none foundin the DSP portion of the molecule.

[0377] 2. Secondary Structure:

[0378] GCG peptide structure prediction profiles ofhydrophobicity/hydrophilicity, antigenicity, flexibility and cellsurface probability are shown in FIGS. 3 to 6. These Figures showGCG-peptide structure prediction analysis of the primary amino acidsequence. Hydrophobicity and hydrophilicity indices are represented astriangles and ovals respectively. Glycosylation motifs are representedas circles on stalks at residues 382-386. Glycosylation symbols can beenseen more clearly in FIG. 6. Protein turn is indicated by the shape ofthe line representing primary amino acid sequence. Regions of (-helix,coil and sheet structure are indicated by localized undulations of theline (refer to FIG. 7 for more detail). Computer predictions were madeusing GCG-software derived from HGMP resource center Cambridge (Rice,1995) Programme Manual for the EGCG package. (Cambridge, CB 10 1 RQ,England: Hinxton Hall). A striking feature is the lack of Sistineresidues and the high degree of hydrophilicity, with four minor siteswith low hydrophobic indices residues 48-53, 59-70, 82-89, and 234-241).The protein does not have a transmembranous profile as deduced from asecondary structure prediction using antheorplot software. The proteinis also highly antigenic and flexible (FIGS. 4 and 5). The overallsecondary structure profile is indicative of an extracellular secretedprotein, and is in agreement with the proposed function of the molecule.FIG. 7 shows the helical, sheet structure, turn and coil regions of thephosphatonin. This is based on a prediction using Gamier analysis of theantheplot v2.5e package. The four lines in each section (top to bottom),represent helix, coil, sheet, and turn probability indices of primaryamino acid sequence. The graph at the bottom presents the same data inblock form. Notable is the high helical content, particularly at the NH2terminus and also towards the C-terminus, which may have a functionalcontext.

EXAMPLE 6 Medical Uses of Phosphatonin and Phosphatonin Fragments

[0379] A number of disorders are amenable to treatment usingpolypeptides according to the present invention.

[0380] X-linked Rickets (hypophosphatemia) (HYP):

[0381] X-linked hypophosphatemic rickets is one of the commonestinherited diseases of bone mineral metabolism (Rowe, 1997). Phosphatoninbioactive fragments such as those cleaved by PHEX and the uncleavedhormone will play a major role in the treatment of the disease. Theprotein cloned and described herein, is predicted to interact with itscognate receptor in the kidney and cause an inhibition in the expressionof a renal Na-dependent phosphate co-transporter (NaPi), and eitherdirectly or indirectly up-regulation of a renal 24 hydroxylase. It isalso predicted to down regulate expression of renal 1 ( hydroxylase(directly/indirectly). After cleavage with PHEX or otherpost-translational modifiers, the peptide fragments derivative of thehormone are predicted to have the opposite bio-function (up-regulationof NaPi, down-regulation of 24 hydroxylase, up regulation of 1 alphahydroxylase). The fragment containing the RGD cell attachment residue(152-154), is predicted to play a role in the receptor interactions,although other peptide derivatives may also mediate receptor ligandinteractions for disparate bioactivities. Also, phosphatonin derivativeswill play an important function in the normalization of thehypomineralised bone lesions. This is predicted to occur by mediatingchanges in the osteoblast mediated mineralization of osteoid, and bycorrecting the aberrant expression/phosphorylation of bone mineralmatrix proteins (osteopontin/osteocalcin). The RGD cell attachmentsequence and also the glycosaminoglycan attachment motif could berequired for the functional nucleation and crystallization ofhydroxyapatite and bone mineral.

[0382] Growth impairment is a major feature of HYP, and currenttreatments are unsuitable. Treatment by administration ofphosphatonin-derived fragments as opposed to inorganic phosphate andvitamin D supplementation, may correct this.

[0383] Accordingly, among the useful effects of peptide fragments ofphosphatonin are:

[0384] 1. Correction of hypophosphatemia (NaPi, preferably renal)

[0385] 2. Normalization of 24-hydroxylase 1 alpha hydroxylase activity(renal).

[0386] 3. mineralization of bone and bone repair (correction/preventionof rickets).

[0387] 4. Complete loss of bone pain symptoms.

[0388] 5. Correction of stunted growth.

[0389] Oncogenic Hypophosphatemic Osteomalacia (OHO):

[0390] The clinical profile of OHO is similar to HYP. There is a renalphosphate leak, low circulating levels of 1,25 dihydroxy vitamin D3(calcitriol), elevated alkaline phosphatase, bone hypomineralizationthat in adults is presented as a generalized bone softening(osteomalacia) and low serum phosphate. The pathophysiologies of HYP andOHO clearly overlap. In rickets, the defect is a non functional PHEXgene. However, in OHO it is circulating unprocessed phosphatonin. Thetumours are often difficult to find, and can be extremely difficult anddangerous to resect. Control of phosphate metabolism and bonemineralization is essential when removal of tumour is contra-indicated.Administration of PHEX to patients to cleave hormone is predicted to bedangerous as other circulating hormones and proteins may also beaffected by promiscuous cleavage. Phosphatonin-fragments could insteadbe designed that have high receptor affinity and bioactivity, such thatthey would compete effectively with unprocessed tumour-derivedcirculating hormone.

[0391] Other Rickets or Hypophosphatemic Conditions:

[0392] There are many causes of rickets besides HYP and OHO, the mostcommon involve abnormalities of vitamin D, but there are causes such ashypophosphatemia, renal tubular acidosis, use of certain medications,sprue, cystic fibrosis etc. Use of fragments of phosphatonin, andphosphatonin itself may be of use in treating these diseases. Some ofthe diseases are briefly discussed below (diseases resulting inhyperphosphatemia are potentially treatable by use of the wholehormone).

[0393] Renal Transplants and Renal Osteodystrophy:

[0394] A chronic feature of renal transplantation is the development ofa renal phosphate leak (hypophosphatemia), and abnormal bonemineralization. Phosphatonin fragments would be effective in treatingthis without the side-effects associated with current medications.

[0395] Osteodystophy (a combination of bone disorders), is usuallycaused by chronic kidney failure (renal disease). Renal failure willresult in death, unless dialysis is given (end stage renal disease).Therefore, patients with osteodystrophy are usually on dialysis therapy.This bone disease, which is also referred to as “renal osteodystrophy”,is common in patients on chronic hemodialysis. Secondaryhyperparathyroidism develops in most patients with chronic renalfailure, and is associated with the histologic finding of osteitisfibrosa cystica. The disease is characterized by growth failure andsevere bone deformities in children, especially the very young. Thepathogenesis of renal osteodystrophy is related to phosphate retention(hyperphosphatemia), and its effect on calcium and calcitriolmetabolism, in addition to roles played by metabolic acidosis,cytokines, and degradation of parathyroid hormone. Treatment includesrestriction of dietary phosphorous intake, phosphate binders, and use ofactive metabolites of vitamin D. In this context addition of unprocessedhormone would be a powerful means of controlling phosphate levels, andwould lead to bone healing. If receptors for phosphatonin are expressedin a range of tissues as well as the kidney, then the potential fortreating patients with end stage renal disease exists (i.e. completeloss of kidney function).

[0396] Osteoporosis/bone Mineral Loss:

[0397] Post-menopausal women are prone to loss of bone mineral withconsequent damage to the integrity of the skeleton. The cause is unknownbut is likely to involve a complex interaction of genetic andenvironmental factors. Current research is focussed on refiningstatistical models to analyze multifactorial diseases such asosteoporosis.

[0398] The use of phosphatonin-derivative fragment(s) would help in thetreatment of this disease by potentially reversing the bone mineralloss. Moreover, the bioactive peptides could be modified to increasepotency and specificity of action.

[0399] Pagets Disease of Bone:

[0400] Pagets disease occurs due to asynchronous bone re-modeling. Bonemineralization (mediated by osteoblasts), and bone resorption (mediatedby osteoclasts), are out of step. Excessive osteoclast resorptiveactivity occurs (predominantly in the early resorptive phase), and bonemarrow is replaced by fibrous tissue and disorganized trabeculae.Although the cause is unknown, administration of peptide derivatives ofphosphatonin may help in the treatment of the disease.

[0401] Diseases Related to Disorders in NaPi in Other Tissues thanKidney:

[0402] The sodium dependent phosphate co-transporter (NaPi) is expressednot just in the kidney but in many other tissues. Three type of NaPi,namely Type I, II, and III have been described thus far and all of themare said to be expressed in the kidney. In tissues other than thekidney, Type III is said to be expressed ubiquitously (Murer, Eur. J.Physiol. 433 (1997) 379-389; Kavanaugh, Kidney Int. 49 (1996) 956-963)and Type I has been confirmed to be expressed in the liver and brain inaddition to the kidney (Hilfiker, PNAS 95 (1998), 14564-14569). On theother hand, Type II had been believed to be expressed only in theproximal tubule of the kidney.

[0403] Although the proximal tubule of the kidney is known to expressall of the above three types, it is widely accepted that Type II playsthe most significant role in terms of phosphate reabsorption at thissite. This has been demonstrated by a knockout mouse in which the gene(named Npt2) encoding Type II NaPi was inactivated. The homozygousmutants (Npt2-/-) exhibited increased urinary phosphate excretion,hypophosphatemia, elevation in the serum concentration of1,25-dihydroxyvitamin D, and other typical symptoms with hereditaryhypophosphatemic rickets with hypercalciuria (HHRH) (Beck, PNAS 95(1998), 5372-5377). Since the regulation of phosphate homeostasis inmammals is largely determined by the kidney, this result is thought todemonstrate that Type II NaPi plays the most important role in systemicphosphate homeostasis among all three types. Also, these facts, togetherwith the result from the CL8 cell line experiment in the examplesindicate that the NaPi that is regulated by Phosphatonin in the kidneyis predominantly the Type II.

[0404] One of the major clinical problems with renal failure patients ishyperphosphatemia. There is a significant clinical value if suchexcessive serum phosphate is controlled. Therefore, phosphatonin, itsfragments or derivatives which can downregulate NaPi and reduce serumphosphate level has a major potential value. In progressive renalfailure patients (before so-called end stage renal disease=ESRD),downregulation of NaPi expressing in the kidney by phosphatonin will bevaluable.

[0405] However, once these patients become ESRD and the majority ofkidney function is lost, phosphatonin will eventually lose its actionsite in the kidney because no more phosphate will be excreted fromglomeruli. At such a disease stage, a potential value exists incontrolling phosphate absorption from the diet in the digestive tract.The digestive tract, particularly the intestine, is the only place wherephosphate is taken up from the diet into the circulation. Therefore,this will be the next major target to control phosphate uptake into thecirculation after the kidney function is lost.

[0406] A subtype of the Type II NaPi, named Type IIb was reported to becloned from mouse intestine (Hilfiker, PNAS 95 (1998), 14564-14569).Although it is yet to be known if phosphatonin can effect on theintestinal Type Ilb NaPi, it is reasonably expected that this Type IlbNaPi in the intestine plays a major role in the absorption of phosphatefrom the diet and that phosphatonin may be the most significant factorfor its up- and downregulation.

EXAMPLE 7 Pharmaceutical Compositions

[0407] Pharmaceutical compositions may be formulated comprising apolypeptide according to the present invention optionally incorporatinga pharmaceutically-acceptable excipient, diluent or carrier. The exactnature and quantities of the components of such compositions may bedetermined empirically and will depend in part upon the route ofadministration of the composition. Routes of administration to patientsinclude oral, buccal, sublingual, topical (including ophthalmic),rectal, vaginal, nasal and parenteral (including intravenous,intraarterial, intramuscular, subcutaneous and intraarticular). In orderto avoid unwanted proteolysis, a parenteral route is preferred.

[0408] Suitable dosages of a molecule of the present invention willvary, depending upon factors such as the disease or disorder to betreated, the route of administration and the age and weight of theindividual to be treated. For instance for parenteral administration, adaily dosage of from 0.1 μg to 1.5 mg/kg of a molecule of the inventionmay be suitable for treating a typical adult. More suitably the dosemight be 1 μg to 150 μg. Accordingly, it is envisaged that the activepolypeptide ingredient may be given in a dose range of from 0.01 to 100mg, typically 0.1 to 10 mg, on a daily basis for an adult human.

[0409] Compositions for parenteral administration for example willusually comprise a solution of the molecule dissolved in an acceptablecarrier, preferably an aqueous carrier. A variety of aqueous carrierscan be used such as water, buffered water, 0.4% saline, 0.3% glycineetc. Such solutions should advantageously be sterile and generally freeof aggregate and other particulate matter. The compositions may containpharmaceutically acceptable buffers to adjust pH, or alter toxicity, forexample sodium acetate, sodium chloride, potassium chloride, calciumchloride, sodium lactate, etc. The concentration of molecule in theseformulations can vary widely, for example from less than about 0.5% toas much as 15 or 20% by weight and could be selected as appropriate by askilled person.

[0410] Typical pharmaceutical compositions are described in detail inRemington's Pharmaceutical Science, 15th ed., Mack Publishing Company,Easton, Pa. (1980). For example, phannaceutical compositions forinjection could be made up to contain 1 ml sterile buffered water, and50 mg of molecule. A typical composition for infusion could be made upto contain 250 ml of sterile Ringer's solution, and 150 mg of molecule.Actual methods for preparing compositions will be known or apparent tothose skilled in the art. Approaches to formulation and administrationof polypeptide pharmaceutical compositions are well-known to thoseskilled in this art and are discussed, for example, by P. Goddard inAdvanced Drug Delivery Reviews, 6 (1991) 103-131.

EXAMPLE 8 Further Characterization of Phosphatonin (MEPE) and itsEncoding Gene

[0411] Clinical Profile of Patients (BD, ND, EM and DS) with OncogenicOsteomalacia:

[0412] Patient BD has been described in an earlier publication (Rowe,Bone 18 (1996), 159-169), and a case report for patient ND has also beenpublished (David, J. Neurosug. 84 (1996), 288-292). Both patientsexhibited classical tumour-osteomalacia, and presented with low serumphosphate and radiological osteomalacia, and low serum 1,25 vitam D3.Patient BD (44 year old woman), and patient ND (66 year old woman),exhibited complete remission of symptoms after removal of tumours fromthe left nasal cavity (haemangiopericytoma), and the intracranial space(mesenchymal hemopericytoma like tumour), respectively. Patient ND hadthree such operations over a period of twenty years, and remissionoccurred after each resection.

[0413] Tumour Conditioned Media:

[0414] Tumour samples from both BD, ND and EM were collected immediatelyafter resection. Samples were then cut into ˜1 mm pieces and some frozenin liquid nitrogen. The remaining pieces of tumour tissue were processedfor tissue culture as described previously (Rowe, Bone 18 (1996),159-169). In brief, samples were digested with collagenase overnight,and then subjected to alternate cycles of culture in the presence andabsence of serum (DMEM media). With patient ND, additional samples from,surrounding sub-dura, and dura were also collected and treated asdescribed above. Also, control skin fibroblast cultures from patient BDwere obtained on the same day as tumour resection, and treated in thesame way as the tumour samples. Samples from patient BD were labeled asfollows: 1: tumour conditioned media (TCM-BD); 2: skin conditioned media(SCM-BD). Samples from patient ND were labeled as follows: 1: Tumourconditioned media (TCM-ND); 2: sub-dura conditioned media (SDCM-ND); 3:dura conditioned media (DCM-ND); 4: fluid surroundingintracranial-tumour (FST-ND). All samples were collected from culturecycles in which cells were grown in serum-free DMEM media, unlessindicated in the text by addition of ‘serum supplemented’ to the aboveabbreviations.

[0415] Concanavilin A Affinity Chromatography of TCM:

[0416] Concanavilin-A affinity chromatography of tumour conditionedmedium (TCM) from patient ND, performed in accordance with Example 1resulted in the isolation of high and low affinity fractions (HCA, andLCA respectively). Both HCA and LCA fractions were eluted withα-methyl-D-glucopyranoside (0.5M) elution buffer. Briefly, partialpurification of TCM proteins was carried out by Conacanavilin A affinitychromatography using a method described by (Wagner, Gen. Comp.Endocrinol. 63 (1986), 481-491), with modifications. Concanavilin ASepharose (Pharmacia Code No: 17-0440-01, 14 ml), in 20% Ethanol, wasfirst washed with several column volumes of water, and then equilibratedin running buffer (CRB; 0.06M Sodium phosphate pH 7.2 and 0.5M NaCl).The equilibrated slurry was then added to a 12 mm×115 mm Pharmacia screwtop column, and three column volumes of CRB running buffer added at aflow rate of 0.4 ml/min (FPLC/HPLC millenium Waters chromatographysystem). Conditioned media equilibrated in CRB buffer (10 ml), was thenadded to the column and allowed to bind. The column was then washed withseveral column volumes of CRB loading buffer, and elutions of boundproteins was then carried out by addition of sodium phosphate elutionbuffer (ERB; 60 mM pH 7.2/0.5M NaCl/0.5Mα-methyl-D-glucopyranoside/0.01% azide), at a flow rate of 0.2 ml/min(40 ml). High affinity proteins were eluted after incubation of thecolumn overnight in ERB buffer followed by a second passage of ERBbuffer at 0.2 ml/min. Elution profiles for both high and lowconcanavilin A-affinity TCM-proteins were identical and produced asingle symmetrical peak at 1.6 column volumes. Peak LCA represented ⅓the total mass of peak HCA, and 1 ug of HCA material was retrieved from10 ml of tumour conditioned media (TCM), from patient ND.

[0417] SDS-PAGE of TCM and Concanavilin A Fractions:

[0418] Tumour conditioned medium, conditioned media and concanavilin Apeaks (HCA and LCA), were separated by SDS-PAGE and visualized afterSybr-Orange staining. SDS-polyacrylamide gel electrophoresis was carriedout using a Novex NuPAGE™ Electrophoresis system consisting of 4-12%Bis-Tris acrylamide-gradient gels (pH 6.4), and MOPS-SDS (50 mM3-[N-morpholino] propane sulfonic acid; 50 mM Tris-base; 3.5 mM SDS; 1.0mM EDTA; pH 7.7) running buffer. Runs were carried out at a constantvoltage of 200 for 50 min. Samples were denatured at 70° C. for 10minutes in NuPage LDS sample buffer (10% glycerol; 1.7% Tris-Base; 1.7%Tris-HCl; 2% Lithium Dodecyl Sulfate; dithiothreitol 50 mM; 0.015% EDTA;0.075% Serva Blue G250; 0.025% Phenol red; pH7.5 final concentration).NuPage antioxidant was added to the upper electrophoresis chamber asrecommended by the manufacturers. Following electrophoresis proteinswere stained by incubating the gels in 7.5% acetic acid supplementedwith SYPRO-Orange. Visualization of proteins was achieved after UVillumination using a Bio-Rad Fluorlmager gel-imaging system. HCA and LCAfractions stained positive for two proteins at 56 kDa and 200 kDarespectively and gave identical profiles. Conditioned media (patientND), from intracranial-tumour, sub-dura (immediately adjacent to tumourin the patient), and dura material contained several major bandsspanning ˜50-80 kDa. A prominent band was present in all preparations at˜66 kDa with a weaker very high molecular weight component at ˜200 kDapresent in tumour and sub-dura. The relative intensity of the ˜200 kDawas highest in the tumour material, and absent in the dura. A diffuseset of bands at ˜55-60 kDa was present in tumour and sub-dura but absentin the dura conditioned media (patient ND). Conditioned media from skinand media control did not reveal any staining for protein. Conditionedmedia from patient BD and EM gave similar profiles except for theabsence of the high molecular weight protein at 200 kDa. Nonphosphaturictumour tissues from patients LA and SL, and also skin controls allcontained the 66 kDa band and also diffuse staining at 50-60 kDa.Concanavilin-A affinity peaks HCA and LCA were enriched for the highmolecular weight 200 kDa band and also contained proteins from the 50-66kDa range. Conditioned media from bone cell lines HTB96 and SaOs2 gavealmost identical protein profiles to tumour conditioned media fromOHO-patient ND. The 200 kDa band intensity in SaOS2 was reduced relativeto TCM from brain tumour (patient ND), sub-dura (patient ND), and CMfrom HTB96.

[0419] Immuno-blotting and Glycoprotein Staining of TCM and PurifiedFractions:

[0420] For western-blotting, proteins were transferred to PVDF membranes(Amersharn), using submarine electrophoresis. After SDS-PAGEelectrophoresis, gels were equilibrated in transfer buffer: 25 mMTris-HCl; 0.38 M glycine; 0.2% SDS (TB) for 1 h at room temperature.PVDF membranes were cut to size, briefly rinsed in methanol, washed indistilled water, and then equilibrated in TB. The equilibrated gel andPVDF membrane were then sandwiched between filters and placed in acassette. The cassette was then placed in a Hoeffer system submarineelectroblotter with TB buffer and cooling maintained at 4° C. bythermocooler. Transfer of proteins was then carried out by positioningthe PVDF end of the sandwich towards the anode, and electrophoresis at aconstant 0.4 A (45V), for 45 min. Blots were screened with 1/1000dilution of pre-Anti-op antisera, post-Anti-op-antisera, or calmodulinconjugated to alkaline phosphatase using the methods described in theEnhanced-Chemiluminescence kit (Amersham; ECL+), or the calmodulinaffinity detection kit (Stratagene) respectively. Chemiluminescence, wasdetected and filmed using the Bio-Rad FluorImaging system, and thecalmodulin-affinity binding was visualized using the colourometricsystem discussed earlier for clone detection (Stratagene). Biotinylatedmolecular weight markers (Amersham), were used as internal controls toasses transfer and molecular weight. Streptavidin conjugated to horseradish peroxidase (HRP), was added to the secondary antibody(goat-anti-rabbit IgG conjugated to HRP), to facilitate visualization ofthe biotinylated-markers via chemiluminescence.

[0421] Western blots of phosphaturic tumour-conditioned-media (TCM),from OHO-patients gave positive chemiluminescent bands when screenedwith pre-absorbed pre-operation antisera. Non-phosphaturic tumours,tissue controls from skin and media controls were all negative whenscreened with pre-absorbed pre-operation antisera. Also, all TCM andconditioned media samples were negative when screened withpost-operation antisera.

[0422] Screening of TCM proteins from patient ND, and osteosarcoma celllines HTB96 and SaOS2 with pre-absorbed pre-operation antiserum revealedtwo distinct immuno-positive bands at ˜54-57 kDa and ˜200 kDa. PatientND tumour sample and adjacent sub-dura tissue gave much stronger 54-57kDa signals relative to dura brain-sample conditioned-media, and nostaining for the 200 kDa band was found in the dura conditioned-media.Both HCA and LCA concanavilin-A fractions contained a very strong signalfor the 200 kDa band, and a reduced but visible signal at 54-57 kDa.Cell lines SaOS2 and HTB96 were also positive for the same bands, butSaOS2 conditioned media had a reduced signal for the 200 kDa bandrelative to TCM and HTB96.

[0423] Skin conditioned media (patient ND and BD), and media controlswere negative, as were screenings with post-operation antisera (Rowe,Bone 18 (1996), 159-169). Recombinant MEPE (rec-MEPE), stainedpositively with pre-absorbed pre-operation antisera, and this could becompeted out with added rec-MEPE). A positive band of 54-57 kDa wasobtained with Sybr-Orange protein stained, and pre-absorbedpre-operation antisera screened rec-MEPE. This was the same size as the55-57 kDa band (pre-absorbed-pre-operation western screened), found withpatient ND tumour conditioned media, and osteosarcoma cell lines HTB96and SaOS2. Recombinant-MEPE contains an additional 4.5 kDa CBP-tag atthe N-terminus that decreases mobility and results in an apparentincrease in molecular weight on SDS-PAGE gels. Thus, the equivalent sizeof tumour derived protein and rec-MEPE may be due to post-translationalmodification of tumour derived MEPE (possibly glycosylation).

[0424] TCM western blots from OHO-tumour patients BD and EM containedmajor pre-absorbed-pre-operation antisera positive bands at slightlylower molecular weight (48-52 kDa), as well as a band co-migrating at55-57 kDa with rec-MEPE. Other higher molecular weight bands were alsoseen at 61, 75, 80, and 93 kDa (weaker signals).

[0425] In all samples the major SYBR-Orange stained protein band at 66kDa was negative when screened with pre-absorbed pre-operation antisera.Glycoprotein screening of duplicate blots gave the same results asscreening with pre-operation antisera and both 54-57 kDa and 200 kDabands stained positive confirming that these proteins are glycosylated.Proteins were separated by SDS-PAGE and blotted onto PVDF membranes asdescribed in methods above. Specific glycoprotein detection was carriedout using an Immuno-Blot kit for glycoprotein detection (Bio-Rad), andAmersham biotinylated markers were added as internal controls. Briefly,after transfer membranes were treated with 10 mM sodium periodate insodium acetate/EDTA buffer to oxidise carbohydrate moieties. The blotswere then washed in PBS and incubated with hydrazide insodium/acetate/EDTA buffer for 60 minutes at room temperature. Filterswere then washed three times (10 minutes) with TBS. Subsequent blockingand detection was carried out as described earlier using the Enhancedchemiluminescence kit (Amersham), and streptavidin horse radishperoxidase. Primary antibody and secondary goat anti-rabbit-HRP was notused.

[0426] In conclusion pre-absorbed pre-operation antisera specificallydetects proteins derived from oncogenic hypophosphatemicosteomalacia-TCM. The major proteins detected fall into two threedistinct molecular size ranges 48-52 kDa, 54-57 kDa, and 200 kDa. AllOHO-TCM samples were positive for the 54-57 kDa protein, and allproteins detected by pre-absorbed-pre-operation antisera stainedpositive when screened for glycoprotein status. Non OHO-tumours controltissues and media were negative when screened with pre-absorbedpre-operation antisera.

EXAMPLE 9 Expression of MEPE Fusion-protein from pCAL-n-EK Vector

[0427] The entire cDNA coding sequence was subcloned into pCAL-n-EK asdescribed in Example 4a. Validation of the fusion construct generated byIPTG induction of the E. coli host BL2 1 (DE3), was achieved byscreening western blots with pre-operation antisera, and also withcalmodulin conjugated to alkaline phosphatase as described above. Thefusion protein with microbial CBP-tag (calmodulin binding peptide of 4.5kDa), containing calmodulin peptide, enterokinase site, and thrombinsite was 56 kDa as deduced by SDS-PAGE. This is in approximate agreementwith the expected molecular size (˜48 kDa). Purification of protein wasachieved by calmodulin affinity chromatography as described above.Preincubation of pre-operation antisera with purified fusion constructresulted in a diminution of the 55-57 kDa signal observed on screeningTCM western blots, but not the 200 kDa band. The failure to completelyreduce the 55-57 kDa signal was presumed to be due to specificrecognition of the highly antigenic glycosylation moiety present in thenascent MEPE-protein (TCM), but absent in the microbial fusion-constructof rec-MEPE. The fusion protein was soluble in aqueous Tris-buffers anddetergents were not required at any stage of the purification process.

EXAMPLE 10 Tissue Expression (RT/PCR and Northern Analysis)

[0428] Northern blots containing poly A+RNA were screened with MEPE cDNAand no hybridization was detected to stomach, thyroid, spinal cord,lymph node, trachea, adrenal gland, bone marrow, heart, brain, lung,liver, skeletal muscle, kidney, and pancreas (Clontech MTN-blots I andIII). For Northern analysis two blots from Clontech (MTN™ and MTN™ III),containing the following poly A+ RNA's: 1; heart, 2; brain, 3; placenta,4; lung, 5; liver, 6; skeletal muscle, 7; kidney, 8; pancreas, 9;stomach, 10; thyroid, 11; spinal cord, 12; lymph node, 13; trachea, 14;adrenal gland, 15; bone marrow, were screened with MEPE cDNA amplifiedwith specific internal primers (Pho433-111F and PHO877-111R). Primersequences for Pho433-111F and PHO877-111R are highlighted in FIG. 8(nucleotide positions 433 to 456 (SEQ ID NO: 24) and 877 to 900 (SEQ IDNO: 25), respectively), and the following PCR conditions were used:predenaturation; 95° C. 3 min; followed by thirty cycles ofdenaturation; 95° C. 45 sec, annealing; 65° C. 30 sec, polymerization;72° C. 45 sec, and a final extension of 72° C. 7 min followed by coolingto 4° C. PCR-buffer (PB), was used with a final concentration of 2 mMMgCl₂. The 444 bp amplified MEPE cDNA product was then resolved bysubmarine agarose electrophoresis, visualized by ethidium bromidestaining, and purified using glass beads (Gene-clean II kit; Bio 101INC). Purified DNA was then radiolabeled using α-P³² dCTP (3000 ci/mmol)in conjunction with the MegaPrime labeling-kit from Amersham. Specificactivities of 5×10⁹ cpm/μg were routinely obtained. Hybridization (60°C.), and prehybridization (60° C.), of blots were carried out usingpublished methods (Rowe, Hum. Genet. 97 (1996), 354-352), and stringencywashes were carried out as follows: 1; two washes at room temperaturefor 30 min with 2× SSC 0.1% SDS, two washes at 60° C. for 30 min in 0.1×SSC 0.1% SDS. Filters were then exposed to film for 7 days at −80 C andthe films developed. Total human-RNA from adrenal glands, brain,duodenum, heart, kidney, liver, lung, skin, spleen, thymus, thyroid,tonsil, did not amplify using RT/PCR and MEPE specific primers, althoughevidence for low level expression using cDNA template was found forbrain, kidney, liver and pancreas. For this experiment, total RNA wasextracted from the following human tissues: 1; Thymus, 2; brain, 3;testis, 4; duodenum, 5; heart, 6; skin, 7; liver, 8; tonsil, 9; spleen,10; thyroid, 11; adrenal, 12; lung, 13; kidney, 14; OHO-tumour tissue,14; Human primary osteoblast. Total RNA from Rat primary osteoblast wasalso obtained. MEPE Internal primers as described above (Pho433-111F andPHO877-111R), were used to copy total RNA using reversetranscriptase-PCR and the Perkin Elmer-Roche RNA PCR kit. Briefly, 1 ugof total RNA was dissolved in 20 μl of 10 mM Tris-HCl (pH 8.3), 50 mMKCl, 5 mM MgCl₂, 1 mM dNTPs, 1 unit/μl ribonuclease inhibitor, 2.5unit/μl MULV reverse transcriptase, 0.75 μM down stream primer(PHO877-111R). The mixture was then incubated at 37° C. for 10 min.Upstream primer (Pho433-111F), dNTPs, MgCl₂, and AmpliTaq DNApolymerase, was then added to give final concentrations of 0.15 μM, 200μM, 2 mM, and 2.5 units/100 μl respectively in a total volume of 100 μl.PCR was then carried out using a Perkin Elmer thennocycler (system9700), set to the following program: predenaturation; 95° C. 3 min;followed by thirty five cycles of denaturation; 95° C. 45 sec,annealing; 65° C. 30 sec, polymerization; 72° C. 45 sec, and a finalextension of 72° C. 7 min followed by cooling to 4° C. Amplifiedproducts were resolved using agarose-gel electrophoresis, and verifiedby southern blotting, and sequencing. Also, a panel of normalized cDNA'sderived from a range of non-OHO tumours (Breast carcinoma, lungcarcinoma I, colon adenocarcinoma I, lung carcinoma II, prostaticadenocarcinoma, colon adenocarcinoma II, ovarian carcinoma, pancreaticcarcinoma; Clontech human-tumour panel #K1422-1) were all negative toMEPE PCR, except for very low level expression in one case of colonadenocarcinoma, ovarian carcinoma, and prostatic carcinoma respectively(detected after southern screening of RT/PCR amplified products withradiolabeled MEPE cDNA). In sharp contrast, RT/PCR using MEPE primersamplified poly A+RNA, from OHO tumours, from four separate patients BD,DM, EM, and DS, indicating high levels of expression (normalized againstglyceraldehyde 3-phosphate dehydrogenase and transferrin). Poly A+ RNAfrom non-phosphaturic tumours and control tissues from OHO-patients(skin and material adjacent to tumours), CL8 human-renal cell line,human primary osteoblast cells (purchased from Clonetics H-OST, seematerials), and poly A+ RNA extracted from a presumed tumour-polyp froma patient with linear sebaceous naevus syndrome (TCM from polyp did notinhibit phosphate uptake in human renal cell line CL8), did not amplifyusing MEPE specific primers. Using Clontech purchased cDNA's derivedfrom heart, brain, placenta, lung, liver, skeletal muscle, kidney, andpancreas (human panel I #K1420-1), as templates for MEPE primer PCR, lowlevel expression was detected in brain, liver, lung and pancreas.Sequencing of the MEPE-primer amplified bands revealed complete homologyto MEPE cDNA and southern screening of the amplified bands with MEPEcDNA confirmed the sequencing results. OHO template poly A+ RNA from allOHO-patients consistently amplified an expected band of 480 bp and alower band of 190 bp. The upper band was confirmed by sequencing andsouthern autoradiography as completely homologous to MEPE sequence, andthe lower band was confirmed as a MEPE-derivative by southern analysis.The lower band did not appear in the low level expression normal-tissuesor non OHOtumours. This indicates that alternative splicing may play arole in the tumour derived MEPE. All RT/PCR and PCR experiments werenormalized against G3PDH and transferrin.

[0429] In summary high level expression of MEPE (as measured by mRNAlevels), was found only in OHO-tumour samples, and evidence for very lowlevel expression (possibly ectopic), was found in brain, liver, kidneyand three out of eleven non-OHO tumours. Eight out of eleven tumourswere negative for MEPE mRNA expression (RT/PCR), and all results werestandardized against GA3PDH and transferrin RT/PCR primers,

EXAMPLE 11 Southern Analysis (Genomic Blots)

[0430] Genomic blots containing immobilized DNA derived from a familywith autosomal rickets (Rowe, Hum. Genet. 91 (1993), 571-575), anddigested with PstI, EcoRI, PvuIl, and MspI respectively were screenedwith radiolabeled MEPE cDNA as described above. Southern analysis wascarried out using genomic digests of DNA extracted from blood asdescribed previously (Rowe, Hum. Genet. 93 (1994), 291-294). The PstIblot revealed the presence of an 11 kb band and also a 4 kb polymorphismin one of the sixteen family members screened. The EcoRI, PvuII, andMspI blots were all positive for single bands of 6 kb, 6.5 kb, and 4 kbrespectively, and confirmed the human provenance of the gene. Due to thelack of genetic information it was not possible to deduce whether thegene segregated with the disease in this autosomal rickets family.

EXAMPLE 12 Phosphate Uptake in a Human Renal Cell Line CL8: TCM and MEPESupplementation

[0431] Phosphate and glucose uptake experiments were conducted on ahuman renal cell line (CL8) as described previously (Rowe, Bone 18(1996), 159-169). In brief cells were cultured in defined medium (DM),to confluency or overnight incubation in 24 well flat bottom tissueculture plates (Falcon 3047). The DM was then replaced with fresh DMsupplemented with purified fusion protein or concanavilin affinitypurified TCM and left overnight at 37° C. Uptake of P³² and C¹⁴methyl-glucose was then measured (Rowe, Bone 18 (1996), 159-169).

[0432] Addition of TCM (1/20 dilution), to human renal cell linesresulted in a significant reduction in Na+ dependent phosphate uptake asreported earlier (Rowe, Bone 18 (1996), 159-169). This inhibition wasprevented by preincubation of TCM with pre-operation and not postoperation antisera, also reported earlier (Rowe, Bone 18 (1996),159-169). Addition of high and low affinity concanavilin-A purifiedfractions (HCA and LCA respectively), at concentrations of 40 ng/ml alsoresulted in inhibition of Na+ dependent phosphate uptake (NaPi). In bothTCM and concanavilin-A fractions the inhibition was specific tophosphate uptake, and did not affect of Na+ dependent a-methyl-D-glucoseuptake. In all cases the affects were dose dependent.

[0433] Similar experiments were carried out with MEPE fusion-proteinpurified by calmodulin affinity chromatography. Surprisingly,recombinant MEPE did not inhibit Na+ dependent phosphate co-transport,but increased phosphate uptake in a dose dependent manner (see FIG. 24).A doubling of phosphate uptake was observed at 1000 ng/ml (p<0.001).These experiments confirm that MEPE fusion protein specificallyincreases Na+ dependent phosphate co-transport in a human renal cellline CL8.

EXAMPLE 13 Cloning of the cDNA Encoding the N-terminus of thePhosphatonin Protein

[0434] The λZAP II uni library described in Example 4 was used forcloning of a cDNA fragment encoding the additional N-terminus of thephosphatonin protein. 5 μl of the λZAP II library described in Example44 was used as substrate (a total of 100000 plaque forming units) in atotal volume of 50 μl consisting of the following ingredients: 10 mMTris-HCL pH 8, 50 mM KCL, 200 μM dNTP's, 2.5 mM MgCl₂, 1 μM GSP2 primer(5′ TCTTCCCCCAGGAGTTTAATC 3′ SEQ ID NO: 28), 1 (M T3 λZAP II specificprimer (5′ ggc cgc aat taa ccc tca cta aag g 3′ SEQ ID NO: 29) and 2.5Units of Promega Taq polymerase. PCR amplification/thermo-cycling was asfollows: 2 min 94° C., 30 sec 94° C., 1 min 63° C., 50 sec 72° C., for30 cycles followed by 10 min at 72° C. and cooling to 4° C. A Gene-AmpPerkin Elmer cycler 9700 was used. The amplified PCR product was thenseparated using conventional submarine Nusieve-agarose electrophoresisin Tris acetate buffer. The DNA-band was then visualized by uv, andligated directly to TA vector pCR2.1 from Invitrogen (using Invitrogensmethod). The ligated molecule was then transformed into E. coli XL1-bluemrf′ (Stratagene cells), using conventional techniques and competentcells as described by Stratagene. Insert and vector were sequenced usingconventional automated sequencing (ABI), and primers derived from vectorand insert. The insert comprised nucleotides 1 to 549 of SEQ ID NO: 26and thus an overlap of about 215 nucleotides at the 3′ end with the5′end of the nucleotide sequence of FIG. 8 (SEQ ID NO: 1). Takentogether a full-length cDNA encoding the entire phosphatonin moleculeshown in SEQ ID NO: 26 and 27, respectively, have now been established.This was confirmed by primer extension experiments that generated a ˜150bp product using a primer (PEP) directed to the new sequence (5′ CAC ACAGCT TTG CTT AGT TTT CTC 3′ SEQ ID NO: 30) and a 146 bp extension productwas expected. Thus, this is very close to the complete cDNA. The primarystructure of the entire phosphatonin protein including the motifspreviously identified for the partial amino acid sequence in Table 1 aswell as additional ones in the N-terminus of the protein are shown inFIG. 15.

[0435] The new sequence data, for example polynucleotides encoding theN-terminus and optionally the entire phosphatonin molecule and thecomplementary strands thereof as well as oligonucleotide primers havingthe nucleotide sequence of SEQ 28 or 30 are particularly suited foridentifying and isolating genomic clones of phosphatonin includingpromoter sequences from an appropriate library.

[0436] While the present invention has been described with reference tothe specific embodiments it should be understood by those skilled in theart that various changes may be made and equivalents may be substitutedwithout departing from the true scope and spirit of the invention. Inaddition, many modifications may be made to adapt to a particularsituation, material, composition of matter, process step or steps, tothe objective, spirit or scope of the present invention. All suchmodifications are intended to be within the scope of the claims appendedhereto.

[0437] The entire disclosure of each document cited (including patents,patent applications, journal articles, abstracts, laboratory manuals,books, or other disclosures) in the Background of the Invention,Detailed Description, and Examples is hereby incorporated herein byreference. Moreover, the sequence listing is herein incorporated byreference.

1 30 1 1655 DNA Homo sapien 1 gtgaataaag aatatagtat cagtaacaaagagaatactc acaatggcct gaggatgtca 60 atttatccta agtcaactgg gaataaagggtttgaggatg gagatgatgc tatcagcaaa 120 ctacatgacc aagaagaata tggcgcagctctcatcagaa ataacatgca acatataatg 180 gggccagtga ctgcgattaa actcctgggggaagaaaaca aagagaacac acctaggaat 240 gttctaaaca taatcccagc aagtatgaattatgctaaag cacactcgaa ggataaaaag 300 aagcctcaaa gagattccca agcccagaaaagtccagtaa aaagcaaaag cacccatcgt 360 attcaacaca acattgacta cctaaaacatctctcaaaag tcaaaaaaat ccccagtgat 420 tttgaaggca gcggttatac agatcttcaagagagagggg acaatgatat atctcctttc 480 agtggggacg gccaaccttt taaggacattcctggtaaag gagaagctac tggtcctgac 540 ctagaaggca aagatattca aacagggtttgcaggcccaa gtgaagctga gagtactcat 600 cttgacacaa aaaagccagg ttataatgagatcccagaga gagaagaaaa tggtggaaat 660 accattggaa ctagggatga aactgcgaaagaggcagatg ctgttgatgt cagccttgta 720 gagggcagca acgatatcat gggtagtaccaattttaagg agctccctgg aagagaagga 780 aacagagtgg atgctggcag ccaaaatgctcaccaaggga aggttgagtt tcattaccct 840 cctgcaccct caaaagagaa aagaaaagaaggcagtagtg atgcagctga aagtaccaac 900 tataatgaaa ttcctaaaaa tggcaaaggcagtaccagaa agggtgtaga tcattctaat 960 aggaaccaag caaccttaaa tgaaaaacaaaggtttccta gtaagggcaa aagtcagggc 1020 ctgcccattc cttctcgtgg tcttgataatgaaatcaaaa acgaaatgga ttcctttaat 1080 ggccccagtc atgagaatat aataacacatggcagaaaat atcattatgt accccacaga 1140 caaaataatt ctacacggaa taagggtatgccacaaggga aaggctcctg gggtagacaa 1200 ccccattcca acaggaggtt tagttcccgtagaagggatg acagtagtga gtcatctgac 1260 agtggcagtt caagtgagag cgatggtgactagtccacca ggagttccca gcggggtgac 1320 agtctgaaga cctcgtcacc tgtgagttgatgtagaggag agccacctga cagctgacca 1380 ggtgaagaga ggatagagtg aagaactgagtgagccaaga atcctggtct ccttggggga 1440 atttttgcta tcttaatagt cacagtataaaattctatta aaggctataa tgtttttaag 1500 caaaaaaaaa tcattacaga tctatgaaataggtaacatt tgagtaggtg tcatttaaaa 1560 atagttggtg aatgtcacaa atgccttctatgttgtttgc tctgtagaca tgaaaataaa 1620 caatatctct cgatgataaa aaaaaaaaaaaaaaa 1655 2 430 PRT Homo sapien 2 Val Asn Lys Glu Tyr Ser Ile Ser AsnLys Glu Asn Thr His Asn Gly 1 5 10 15 Leu Arg Met Ser Ile Tyr Pro LysSer Thr Gly Asn Lys Gly Phe Glu 20 25 30 Asp Gly Asp Asp Ala Ile Ser LysLeu His Asp Gln Glu Glu Tyr Gly 35 40 45 Ala Ala Leu Ile Arg Asn Asn MetGln His Ile Met Gly Pro Val Thr 50 55 60 Ala Ile Lys Leu Leu Gly Glu GluAsn Lys Glu Asn Thr Pro Arg Asn 65 70 75 80 Val Leu Asn Ile Ile Pro AlaSer Met Asn Tyr Ala Lys Ala His Ser 85 90 95 Lys Asp Lys Lys Lys Pro GlnArg Asp Ser Gln Ala Gln Lys Ser Pro 100 105 110 Val Lys Ser Lys Ser ThrHis Arg Ile Gln His Asn Ile Asp Tyr Leu 115 120 125 Lys His Leu Ser LysVal Lys Lys Ile Pro Ser Asp Phe Glu Gly Ser 130 135 140 Gly Tyr Thr AspLeu Gln Glu Arg Gly Asp Asn Asp Ile Ser Pro Phe 145 150 155 160 Thr GlyPro Asp Leu Glu Gly Lys Asp Ile Gln Thr Gly Phe Ala Gly 165 170 175 SerGly Asp Gly Gln Pro Phe Lys Asp Ile Pro Gly Lys Gly Glu Ala 180 185 190Pro Ser Glu Ala Glu Ser Thr His Leu Asp Thr Lys Lys Pro Gly Tyr 195 200205 Asn Glu Ile Pro Glu Arg Glu Glu Asn Gly Gly Asn Thr Ile Gly Thr 210215 220 Arg Asp Glu Thr Ala Lys Glu Ala Asp Ala Val Asp Val Ser Leu Val225 230 235 240 Glu Gly Ser Asn Asp Ile Met Gly Ser Thr Asn Phe Lys GluLeu Pro 245 250 255 Gly Arg Glu Gly Asn Arg Val Asp Ala Gly Ser Gln AsnAla His Gln 260 265 270 Gly Lys Val Glu Phe His Tyr Pro Pro Ala Pro SerLys Glu Lys Arg 275 280 285 Lys Glu Gly Ser Ser Asp Ala Ala Glu Ser ThrAsn Tyr Asn Glu Ile 290 295 300 Pro Lys Asn Gly Lys Gly Ser Thr Arg LysGly Val Asp His Ser Asn 305 310 315 320 Arg Asn Gln Ala Thr Leu Asn GluLys Gln Arg Phe Pro Ser Lys Gly 325 330 335 Lys Ser Gln Gly Leu Pro IlePro Ser Arg Gly Leu Asp Asn Glu Ile 340 345 350 Lys Asn Glu Met Asp SerPhe Asn Gly Pro Ser His Glu Asn Ile Ile 355 360 365 Thr His Gly Arg LysTyr His Tyr Val Pro His Arg Gln Asn Asn Ser 370 375 380 Thr Arg Asn LysGly Met Pro Gln Gly Lys Gly Ser Trp Gly Arg Gln 385 390 395 400 Pro HisSer Asn Arg Arg Phe Ser Ser Arg Arg Arg Asp Asp Ser Ser 405 410 415 GluSer Ser Asp Ser Gly Ser Ser Ser Glu Ser Asp Gly Asp 420 425 430 3 4 PRTArtificial Sequence synthesized peptide 3 Ser Gly Asp Gly 1 4 7 PRTArtificial Sequence synthesized peptide 4 Ala Asp Ala Val Asp Val Ser 15 5 22 PRT Artificial Sequence synthesized peptide 5 Ser Ser Arg Arg ArgAsp Asp Ser Ser Glu Ser Ser Asp Ser Gly Ser 1 5 10 15 Ser Ser Glu SerAsp Gly 20 6 21 PRT Artificial Sequence synthesized peptide 6 Ser SerArg Ser Lys Glu Asp Ser Asn Ser Thr Glu Ser Lys Ser Ser 1 5 10 15 SerGlu Glu Asp Gly 20 7 28 PRT Artificial Sequence chemical synthesis inlaboratory 7 Asp Ser Ser Glu Ser Ser Asp Ser Gly Ser Ser Ser Glu Ser AspSer 1 5 10 15 Ser Glu Ser Ser Asp Ser Gly Ser Ser Ser Glu Ser 20 25 8 38DNA Artificial Sequence chemical synthesis in laboratory 8 gacgacgacaaggtgaataa agaatatagt atcagtaa 38 9 35 DNA Artificial Sequencenucleotide sequence 9 ggaacaagac ccgtctagtc accatcgctc tcact 35 10 15PRT Artificial Sequence synthesized peptide 10 Asp Asp Ser Ser Glu SerSer Asp Ser Gly Ser Ser Ser Glu Ser 1 5 10 15 11 16 PRT ArtificialSequence synthesized peptide 11 Asp Asp Ser Ser Glu Ser Ser Asp Ser GlySer Ser Ser Glu Ser Asp 1 5 10 15 12 22 PRT Artificial Sequencesynthesized peptide 12 Ser Ser Arg Arg Arg Asp Asp Ser Ser Glu Ser SerAsp Ser Gly Ser 1 5 10 15 Ser Ser Glu Ser Asp Gly 20 13 14 PRTArtificial Sequence synthesized peptide 13 Asp Ser Ser Asp Ser Ser AspSer Ser Ser Ser Ser Asp Ser 1 5 10 14 15 PRT Artificial Sequencesynthesized peptide 14 Asp Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp SerSer Asp Ser 1 5 10 15 15 14 PRT Artificial Sequence synthesized peptide15 Asp Ser Ser Asp Ser Ser Asp Ser Asn Ser Ser Ser Asp Ser 1 5 10 16 14PRT Artificial Sequence synthesized peptide 16 Asp Ser Ser Glu Ser SerAsp Ser Ser Asn Ser Ser Asp Ser 1 5 10 17 14 PRT Artificial Sequencesynthesized peptide 17 Asp Ser Ser Asp Ser Ser Asp Ser Ser Asn Ser SerAsp Ser 1 5 10 18 16 PRT Artificial Sequence synthesized peptide 18 AspAsp Ser His Gln Ser Asp Glu Ser His His Ser Asp Glu Ser Asp 1 5 10 15 1911 PRT Artificial Sequence synthesized peptide 19 Ser Asp Glu Ser HisHis Ser Asp Glu Ser Asp 1 5 10 20 11 PRT Artificial Sequence synthesizedpeptide 20 Ser Asp Ser Ser Ser Ser Ser Asp Ser Ser Asp 1 5 10 21 11 PRTArtificial Sequence synthesized peptide 21 Ser Asp Ser Ser Asp Ser SerAsp Ser Ser Asp 1 5 10 22 11 PRT Artificial Sequence synthesized peptide22 Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asn 1 5 10 23 21 PRTArtificial Sequence synthesized peptide 23 Ser Ser Arg Ser Lys Glu AspSer Asn Ser Thr Glu Ser Lys Ser Ser 1 5 10 15 Ser Glu Glu Asp Gly 20 2425 DNA Artificial Sequence synthesized peptide 24 ggttatacag atcttcaagagagag 25 25 24 DNA Artificial Sequence synthesized peptide 25 gttggtactttcagctgcat cact 24 26 2013 DNA Homo sapien 26 cacacagctt tgcttagttttctccggcac gagcaggtat tctgaaggtg aaagatacca 60 gagattctca aagatgcgagttttctgtgt gggactactc cttttcagtg tgacctgggc 120 agcaccaaca tttcaaccacagactgagaa aactaagcaa agctgtgtgg aagagcagag 180 gcaggaagaa aaaaacaaagacaatattgg ttttcaccat ttgggcaaga gaataaatca 240 agagctatca tctaaagaaaatattgtcca ggaaagaaag aaagatttgt ccctttctga 300 agccagtgag aataagggaagtagtaaatc tcaaaattat ttcacaaata gacagagact 360 gaataaagaa tatagtatcagtaacaaaga gaatactcac aatggcctga ggatgtcaat 420 ttatcctaag tcaactgggaataaagggtt tgaggatgga gatgatgcta tcagcaaact 480 acatgaccaa gaagaatatggcgcagctct catcagaaat aacatgcaac atataatggg 540 gccagtgact gcgattaaactcctggggga agaaaacaaa gagaacacac ctaggaatgt 600 tctaaacata atcccagcaagtatgaatta tgctaaagca cactcgaagg ataaaaagaa 660 gcctcaaaga gattcccaagcccagaaaag tccagtaaaa agcaaaagca cccatcgtat 720 tcaacacaac attgactacctaaaacatct ctcaaaagtc aaaaaaatcc ccagtgattt 780 tgaaggcagc ggttatacagatcttcaaga gagaggggac aatgatatat ctcctttcag 840 tggggacggc caaccttttaaggacattcc tggtaaagga gaagctactg gtcctgacct 900 agaaggcaaa gatattcaaacagggtttgc aggcccaagt gaagctgaga gtactcatct 960 tgacacaaaa aagccaggttataatgagat cccagagaga gaagaaaatg gtggaaatac 1020 cattggaact agggatgaaactgcgaaaga ggcagatgct gttgatgtca gccttgtaga 1080 gggcagcaac gatatcatgggtagtaccaa ttttaaggag ctccctggaa gagaaggaaa 1140 cagagtggat gctggcagccaaaatgctca ccaagggaag gttgagtttc attaccctcc 1200 tgcaccctca aaagagaaaagaaaagaagg cagtagtgat gcagctgaaa gtaccaacta 1260 taatgaaatt cctaaaaatggcaaaggcag taccagaaag ggtgtagatc attctaatag 1320 gaaccaagca accttaaatgaaaaacaaag gtttcctagt aagggcaaaa gtcagggcct 1380 gcccattcct tctcgtggtcttgataatga aatcaaaaac gaaatggatt cctttaatgg 1440 ccccagtcat gagaatataataacacatgg cagaaaatat cattatgtac cccacagaca 1500 aaataattct acacggaataagggtatgcc acaagggaaa ggctcctggg gtagacaacc 1560 ccattccaac aggaggtttagttcccgtag aagggatgac agtagtgagt catctgacag 1620 tggcagttca agtgagagcgatggtgacta gtccaccagg agttcccagc ggggtgacag 1680 tctgaagacc tcgtcacctgtgagttgatg tagaggagag ccacctgaca gctgaccagg 1740 tgaagagagg atagagtgaagaactgagtg agccaagaat cctggtctcc ttgggggaat 1800 ttttgctatc ttaatagtcacagtataaaa ttctattaaa ggctataatg tttttaagca 1860 aaaaaaaatc attacagatctatgaaatag gtaacatttg agtaggtgtc atttaaaaat 1920 agttggtgaa tgtcacaaatgccttctatg ttgtttgctc tgtagacatg aaaataaaca 1980 atatctctcg atgataaaaaaaaaaaaaaa aaa 2013 27 525 PRT Homo sapien 27 Met Arg Val Phe Cys ValGly Leu Leu Leu Phe Ser Val Thr Trp Ala 1 5 10 15 Ala Pro Thr Phe GlnPro Gln Thr Glu Lys Thr Lys Gln Ser Cys Val 20 25 30 Glu Glu Gln Arg GlnGlu Glu Lys Asn Lys Asp Asn Ile Gly Phe His 35 40 45 His Leu Gly Lys ArgIle Asn Gln Glu Leu Ser Ser Lys Glu Asn Ile 50 55 60 Val Gln Glu Arg LysLys Asp Leu Ser Leu Ser Glu Ala Ser Glu Asn 65 70 75 80 Lys Gly Ser SerLys Ser Gln Asn Tyr Phe Thr Asn Arg Gln Arg Leu 85 90 95 Asn Lys Glu TyrSer Ile Ser Asn Lys Glu Asn Thr His Asn Gly Leu 100 105 110 Arg Met SerIle Tyr Pro Lys Ser Thr Gly Asn Lys Gly Phe Glu Asp 115 120 125 Gly AspAsp Ala Ile Ser Lys Leu His Asp Gln Glu Glu Tyr Gly Ala 130 135 140 AlaLeu Ile Arg Asn Asn Met Gln His Ile Met Gly Pro Val Thr Ala 145 150 155160 Ile Lys Leu Leu Gly Glu Glu Asn Lys Glu Asn Thr Pro Arg Asn Val 165170 175 Leu Asn Ile Ile Pro Ala Ser Met Asn Tyr Ala Lys Ala His Ser Lys180 185 190 Asp Lys Lys Lys Pro Gln Arg Asp Ser Gln Ala Gln Lys Ser ProVal 195 200 205 Lys Ser Lys Ser Thr His Arg Ile Gln His Asn Ile Asp TyrLeu Lys 210 215 220 His Leu Ser Lys Val Lys Lys Ile Pro Ser Asp Phe GluGly Ser Gly 225 230 235 240 Tyr Thr Asp Leu Gln Glu Arg Gly Asp Asn AspIle Ser Pro Phe Ser 245 250 255 Gly Asp Gly Gln Pro Phe Lys Asp Ile ProGly Lys Gly Glu Ala Thr 260 265 270 Gly Pro Asp Leu Glu Gly Lys Asp IleGln Thr Gly Phe Ala Gly Pro 275 280 285 Ser Glu Ala Glu Ser Thr His LeuAsp Thr Lys Lys Pro Gly Tyr Asn 290 295 300 Glu Ile Pro Glu Arg Glu GluAsn Gly Gly Asn Thr Ile Gly Thr Arg 305 310 315 320 Asp Glu Thr Ala LysGlu Ala Asp Ala Val Asp Val Ser Leu Val Glu 325 330 335 Gly Ser Asn AspIle Met Gly Ser Thr Asn Phe Lys Glu Leu Pro Gly 340 345 350 Arg Glu GlyAsn Arg Val Asp Ala Gly Ser Gln Asn Ala His Gln Gly 355 360 365 Lys ValGlu Phe His Tyr Pro Pro Ala Pro Ser Lys Glu Lys Arg Lys 370 375 380 GluGly Ser Ser Asp Ala Ala Glu Ser Thr Asn Tyr Asn Glu Ile Pro 385 390 395400 Lys Asn Gly Lys Gly Ser Thr Arg Lys Gly Val Asp His Ser Asn Arg 405410 415 Asn Gln Ala Thr Leu Asn Glu Lys Gln Arg Phe Pro Ser Lys Gly Lys420 425 430 Ser Gln Gly Leu Pro Ile Pro Ser Arg Gly Leu Asp Asn Glu IleLys 435 440 445 Asn Glu Met Asp Ser Phe Asn Gly Pro Ser His Glu Asn IleIle Thr 450 455 460 His Gly Arg Lys Tyr His Tyr Val Pro His Arg Gln AsnAsn Ser Thr 465 470 475 480 Arg Asn Lys Gly Met Pro Gln Gly Lys Gly SerTrp Gly Arg Gln Pro 485 490 495 His Ser Asn Arg Arg Phe Ser Ser Arg ArgArg Asp Asp Ser Ser Glu 500 505 510 Ser Ser Asp Ser Gly Ser Ser Ser GluSer Asp Gly Asp 515 520 525 28 21 DNA Artificial Sequence synthesizedpeptide 28 tcttccccca ggagtttaat c 21 29 25 DNA Artificial Sequencesynthesized peptide 29 ggccgcaatt aaccctcact aaagg 25 30 24 DNAArtificial Sequence synthesized peptide 30 cacacagctt tgcttagttt tctc 24

1. An isolated or substantially pure form of a polypeptide havingphosphatonin activity.
 2. The polypeptide of claim 1, which has anapproximate molecular weight of 53 to 60 kDa as measured on SDS-PAGE. 3.The polypeptide of claim 1, which has an approximate molecular weight of200 kDa as measured on bis- tris SDS-PAGE at pH
 7. 4. The polypeptide ofany one of claims 1 to 3, which is glycosylated and/or phosphorylated.5. The polypeptide of any one of claims 1 to 4, which is obtainablefollowing purification from Saos-2 cells (Deposit No. ECACC 89050205).6. The polypeptide of any one of claims 1 to 5 or an immunologicallyand/or biologically active fragment thereof, which comprises an aminoacid sequence encodable by a polynucleotide selected from the groupconsisting of (a) polynucleotides encoding at least the mature form ofthe polypeptide comprising the amino acid sequence depicted in SEQ IDNO: 2 (FIG. 8) or SEQ ID NO: 27; (b) polynucleotides comprising thecoding sequence as depicted in SEQ ID NO: 1 (FIG. 8) or SEQ ID NO: 26encoding at least the mature form of the polypeptide; (c)polynucleotides comprising at least a nucleotide sequence encoding aminoacid residues 1 to 96, 18 to 96 or 47 to 96 of SEQ ID NO: 27; (d)polynucleotides encoding a polypeptide derived from the polypeptideencoded by a polynucleotide of (a), (b) or (c) by way of substitution,deletion and/or addition of one or several amino acids of the amino acidsequence encoded by the polynucleotide of (a), (b) or (c); (e)polynucleotides comprising the complementary strand which hybridizeswith a polynucleotide of any one of (a) to (d); (f) polynucleotidesencoding a polypeptide the sequence of which has an identity of 60% ormore to the amino acid sequence of the polypeptide encoded by apolynucleotide of any one of (a) to (e); (g) polynucleotides encoding apolypeptide capable of regulating phosphate metabolism comprising afragment or an epitope-bearing portion of a polypeptide encoded by apolynucleotide of any one of (a) to (f); (h) polynucleotides encoding anepitope-bearing portion of a phosphatonin polypeptide comprising aminoacid residues from about 1 to 40, 141 to 180 and/or 401 to 429 in SEQ IDNO: 2 (FIG. 8) and/or amino acid residues from about 42 to 51 and/or 47to 96 of SEQ ID NO: 27; (i) polynucleotides comprising at least 15nucleotides of a polynucleotide of any one of (a) to (h) and encoding apolypeptide capable of regulating phosphate metabolism; (j)polynucleotides encoding a polypeptide capable of regulating phosphatemetabolism comprising the cell and/or glycosaminoglycan attachment motifand/or the bone mineral motif of a polypeptide encoded by apolynucleotide of any one of (a) to (i); (k) polynucleofides of any oneof (a) to (j) wherein the encoded polypeptide is capable of forming ahomo- and/or heterodimer; and (l) polynucleotides the nucleotidesequence of which is degenerate as a result of the genetic code to anucleotide sequence of a polynucleotide of any of (a) to (k).
 7. Thepolypeptide of any one of claims 1 to 6, which is capable of regulatingphosphate metabolism.
 8. An isolated polynucleotide encoding apolypeptide of any one of claims 1 to
 7. 9. The polynucleotide of claim8, which comprises RNA or DNA.
 10. The polynucleotide of claim 8 or 9,wherein the nucleotide sequence comprises sequential nucleotidedeletions from either the C-terminus or the N-terminus.
 11. Apolynucleotide which hybridizes with the polynucleotide of any one ofclaims 8 to 10 and which encodes a mutated version of the polypeptide ofany one of claims 1 to 7 which has lost at least part of itsphosphatonin activity.
 12. A vector containing the polynucleotide of anyone of claims 8 to
 11. 13. A host cell genetically engineered with thepolynucleotide of any one of claims 8 to 11, the vector of claim 12 orproduced by introducing a expression control sequence into a host cellwhich mediates the expression of a gene encoding the polypeptide of anyone of claims 1 to
 7. 14. A process for isolating a phosphatoninpolypeptide comprising the steps of: (a) culturing tumor-conditionedmedia or osteosarcoma cells to confluence in serum supplemented media(DMEM Eagles/10% FCS/glutamine/antimycotic (DMFCS); (b) incubating thecells on alternate days in serum free media DMEMEagles/glutamine/antimycotic antibiotic (DM) up to five hours; (c)collecting conditioned serum free media from the cells and equilibratingthe conditioned media to 0.06M sodium phosphate pH 7.2 and 0.5 M NaCl(PBS); (d) subjecting the media from (c) to an equilibrated column ofconcanavilin A sepharose; (e) washing the column extensively with PBS;(f) eluting the concanavalin A column with PBS supplemented with 0.5 Mα-methyl-D-glucopyranoside; (g) subjecting the eluted material from (f)to cation exchange chromatography; and (h) eluting phosphatoninpolypeptide containing fractions with 0.5 M NaCl.
 15. A process forproducing a polypeptide having the biological and/or immunologicalactivity of phosphatonin comprising: culturing the host cell of claim 13and recovering the polypeptide encoded by said polynucleotide from theculture.
 16. A polypeptide which is obtainable by the process of claim14 or 15 or by proteolytic cleavage of a phosphatonin polypeptide of anyone of claims 1 to 7 or obtainable by the process of claim 14 or 15 by aZinc-metalloendopeptidase.
 17. The polypeptide of claim 16 which forms ahomo- or heterodimer.
 18. The polypeptide of claim 16, wherein one orboth cysteine residues corresponding to amino acid positions 5 and 31 inSEQ ID NO: 27 are deleted, substituted and/or blocked.
 19. Thepolypeptide of any one of claims 1 to 7, 16 or 17 having at least one ofthe following activities: (a) it is capable of down-regulating sodiumdependent phosphate co-transport; (b) it is capable of up-regulatingrenal 25-hydroxy vitamin D3-24-hydroxylase; and/or (c) it is capable ofdown-regulating renal 25-hydroxy-D-1-α-hydroxylase.
 20. The polypeptideof any one of claims 1 to 7, 16 or 18 having at least one of thefollowing activities: (a) it is capable of up-regulating sodiumdependent phosphate co-transport; (b) it is capable of down-regulatingrenal 25-hydroxy vitamin D3-24-hydroxylase; and/or (c) it is capable ofup-regulating renal 25-hydroxy-D-1-(-hydroxylase.
 21. The polypeptide ofany one of claims 16 to 18 which has lost at least one of the activitiesas defined in claims 19 or
 20. 22. An isolated antibody that bindsspecifically to the isolated polypeptide of any one of claims 1 to 7 or16 to
 21. 23. A nucleic acid molecule of at least 15 nucleotides inlength hybridizing specifically with a polynucleotide of any one ofclaims 8 to 11 or with a complementary strand thereof.
 24. An isolatedregulatory sequence of a promoter regulating the expression of a nucleicacid molecule comprising a polynucleotide of any one of claims 8 to 11.25. A recombinant DNA molecule comprising the regulatory sequence ofclaim
 24. 26. A method for treating a medical condition related to adisorder of phosphate metabolism which comprises administering to amammalian subject a therapeutically effective amount of the polypeptideof any one of claims 1 to 7 or 16 to 21 or of the polynucleotide of anyone of claims 8 to 11, the vector of claim 12 or of the antibody ofclaim
 22. 27. A method of diagnosing a pathological condition or asusceptibility to a pathological condition in a subject related to adisorder of phosphate metabolism comprising: (a) determining thepresence or absence of a mutation in the polynucleotide of any one ofclaims 8 to 11; and (b) diagnosing a pathological condition or asusceptibility to a pathological condition based on the presence orabsence of said mutation.
 28. A method of diagnosing a pathologicalcondition or a susceptibility to a pathological condition in a subjectrelated to a disorder of phosphate metabolism comprising: (a)determining the presence or amount of expression of the polypeptide ofany one of claims 1 to 7 or 16 to 21 in a biological sample; and (b)diagnosing a pathological condition or a susceptibility to apathological condition based on the presence or amount of expression ofthe polypeptide.
 29. A method for identifying a binding partner to aphosphatonin polypeptide comprising: (a) contacting a polypeptide of anyone of claims 1 to 7 or 16 to 21 with a compound to be screened; and (b)determining whether the compound effects an activity of the polypeptide.30. A method of identifying and obtaining a drug candidate for therapyof disorders in phosphate metabolism comprising the steps of (a)contacting the polypeptide of any one claims 16 to 21 or a cellexpressing said polypeptide in the presence of components capable ofproviding a detectable signal in response to phosphate uptake, with saiddrug candidate to be screened under conditions to permit phosphatemetabolism, and (b) detecting presence or absence of a signal orincrease of the signal generated from phosphate metabolism, wherein thepresence or increase of the signal is indicative for a putative drug.31. A method of producing a therapeutic agent comprising the steps ofthe method of any one of claims 28 to 30; and (i) synthesizing thecompound obtained or identified in step (b) or an analog or derivativethereof in an amount sufficient to provide said agent in atherapeutically effective amount to a patient; and/or (ii) combining thecompound obtained or identified in step (b) or an analog or derivativethereof with a pharmaceutically acceptable carrier
 32. Anactivator/agonist or inhibitor/antagonist of phosphate metabolism orbinding partner of phosphatonin obtained by the method of any one ofclaims 28 to
 30. 33. A composition comprising a polypeptide of any oneof claims 1 to 7, or 16 to 21, the polynucleotide of any one of claims 8to 11, a vector of claim 12, an antibody of claim 22, the nucleic acidmolecule of claim 23 or the activator/agonist, inhibitor/antagonist orbinding partner of claim
 32. 34. The composition of claim 33 which is apharmaceutical composition and further comprises a pharmaceuticallyacceptable excipient, diluent or carrier.
 35. The composition of claim34 which is a diagnostic composition and further comprises means fordetection.
 36. Use of a polypeptide of any one of claims 1 to 7 or 16 to21 or a DNA encoding and capable expressing said polypeptide or theactivator/agonist, binding partner of claim 32 or the antibody of claim22, for the preparation of a medicament for treatment of a disorder ofphosphate metabolism.
 37. Use ofapolypeptideofanyoneofclaims 1 to 7 or16, 17, 19 or 21 or a DNA encoding and capable expressing saidpolypeptide, the activator/agonist or binding partner of claim 32 or theantibody of claim 22, for the preparation of a medicament for thetreatment of hyperphosphatemia.
 38. Use of a polypeptide of any one ofclaims 1 to 7, 16, 17, 19 or 21 or a DNA encoding and capable expressingsaid polypeptide or the activator/agonist, binding partner of claim 32or the antibody of claim 22, for the preparation of a medicament for thetreatment of renal osteodystrophy, hyperphophatemia in renaldialysis/pre-dialysis, secondary hyperparathyrodism or osteitis fibrosacystica.
 39. Use of a polypeptide of any one of claims 1 to 7, 16, 18,20 or 21 or a DNA encoding and capable expressing said polypeptide, theantibody of claim 22, the nucleic acid molecule of claim 23 or theinhibitor/antagonist of claim 32, for the preparation of a medicamentfor the treatment of hypophosphatemia.
 40. Use of a polypeptide of anyone of claims 1 to 7, 16, 18, 20 or 21, or a DNA encoding and capableexpressing said polypeptide, the antibody of claim 22, the nucleic acidmolecule of claim 23 or the inhibitor/antagonist of claim 32, for thepreparation of a medicament for the treatment of X-linkedhypophosphatemic rickets, hereditary hypophosphatemic rickets withhypercalcuria (HHRH), hypomineralised bone lesions, stunted growth injuveniles, oncogenic hypophosphatemic osteomalacia, renal phosphateleakage, renal osteodystrophy, osteoporosis, vitamin D resistantrickets, end organ resistance, renal Fanconi syndrome, autosomalrickets, Paget's disease, kidney failure, renal tubular acidosis, cysticfibrosis or sprue.
 41. Use ofa polypeptide ofany one ofclaims 1 to 7,16, 18, 20 or 21, or a DNA encoding and capable expressing saidpolypeptide, the antibody of claim 22, the nucleic acid molecule ofclaim 23 or the inhibitor/antagonist of claim 32, for the manufacture ofa medicament for the treatment of a bone mineral loss disorder.
 42. Useof a polypeptide of any one of claims 1 to 7, 16, 18, 20 or 21 andZinc-metalloendopeptidase for the manufacture of a combined preparationfor simultaneous, separate or sequential use for the treatment of adisorder of phosphate metabolism.
 43. Use of a transformed osteoblast orbone cell line capable of phosphatonin overexpression for the productionof phosphatonin.